McGRAW-HILL  PUBLICATIONS  IN  THE 
AGRICULTURAL  AND  BOTANICAL  SCIENCES 
EDMUND  W.  SINNOTT,  Consulting  Editor 


TEXTBOOK  OF 

AGRICULTURAL  BACTERIOLOGY 


McGRAW-HILL  PUBLICATIONS  IN  THE 
AGRICULTURAL  AND  BOTANICAL  SCIENCES 

Edmund  W.  Sinnott,  Consulting  Editor 


Adams — Farm  Management 

Babcock  and  Clausen — Genetics  in 
Relation  to  Agriculture 

Babcock  and  Collins — Genetics 
Laboratory  Manual 

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Products 

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Conard — Plant  Sociology 

Brown — Cotton 

Carrier — Beginnings  of  Agricul- 
ture in  America 

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Vegetable  Products 

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Manual  of  Fruit  and  Vegetable 
Products 

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duction to  Plant  Anatomy 

Eckles,  Combs  and  Macy — Milk 
and  Milk  Products 

Emerson — Soil  Characteristics 

Fawcett  and  Lee — Citrus  Diseases 
and  Their  Control 

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Phycomycetes 

Gardner , Bradford  and  Hooker — 
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duction 

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tive Morphology  of  Fungi 

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Horlacher — Sheep  Production 
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Loeb — Regeneration 

LOhnis  and  Fred — Textbook  of 
Agricultural  Bacteriology 
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Maximov — A Textbook  of  Plant 
Physiology 

Miller — Plant  Physiology 
Piper  and  Morse — The  Soybean 
Pool — Flowers  and  Flowering 

Plants 

Rice — The  Breeding  and  Improve- 
ment of  Farm  Animals 
Sharp — Introduction  to  Cytology 
Sinnott — Botany:  Principles  and 

Problems 

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Genetics 

Smith — Fresh-water  Algae  of  the 
United  States 

Swingle — A Textbook  of  System- 
atic Botany 

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Life 

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Waite — Poultry  Science  and 
Practice 

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opment of  Vegetable  Crops 
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ogy 


There  is  also  a series  of  McGraw-Hill  Publications  in  the  Zoological 
Sciences,  of  which  A.  Franklin  Shull  is  Consulting  Editor. 


TEXTBOOK  OF 
AGRICULTURAL  BACTERIOLOGY 


BY 

F.  LOHNIS,  Ph.D. 

BACTERIOLOGIST,  UNITED  STATES  DEPARTMENT  OF  AGRICULTURE 
AND 

E.  B.  FRED,  Ph.D. 

PROFESSOR  OF  BACTERIOLOGY,  UNIVERSITY  OF  WISCONSIN 


First  Edition 
Fourth  Impression 


McGRAW-HILL  BOOK  COMPANY,  Inc. 

NEW  YORK  AND  LONDON 
1923 


Copyright,  1923,  by  the 
McGraw-Hill  Book  Company,  Inc. 

PRINTED  IN  THE  UNITED  STATES  OF  AMERICA 


PRESS  OF 

BRAUNWORTH  A CO. 
BOOK  MANUFACTURERS 
BROOKLYN,  Xs 


KNNRMINr  DEPT..  LIBRARY 


. c\  ’b 
U9>  ^ 51 


PREFACE 

This  “Textbook  of  Agricultural  Bacteriology”  was  written  to  give 
the  reader  an  accurate  and  fairly  complete  view  of  this  new  and  wide 
field  of  knowledge.  Most  of  the  material  presented  in  the  book  was 
collected  and  used  by  the  senior  author  while  teaching  at  the  University 
of  Leipzig  (1903-1914),  and  under  the  title  “Vorlesungen  fiber  land- 
wirtschaftliche  Bakteriologie”  was  published  in  1913.  Several  requests 
for  an  English  translation  have  been  received  since  then.  It  was 
deemed  preferable,  however,  to  await  an  opportunity  when  the  whole 
matter  could  be  thoroughly  revised  and  rearranged  in  such  a manner 
as  to  make  the  book  most  useful  for  the  American  and  British  student. 
The  junior  author  has  used  the  “Vorlesungen”  for  his  course  at  the 
University  of  Wisconsin,  while  the  senior  author’s  time  since  1914 
has  been  devoted  exclusively  to  research  work.  It  is  hoped  that  all 
the  varied  experiences  incorporated  in  the  book  will  have  added  to 
its  usefulness.  Inevitably  in  a joint  work  of  this  character  there  were 
differences  of  opinion  between  the  authors  on  certain  points.  Inasmuch, 
however,  as  the  book  was  designed  as  a text,  it  was  felt  that  it  would 
be  inadvisable  to  introduce  any  evidences  of  differences  of  opinion, 
either  by  footnote  or  individual  statements. 

The  first  half  of  the  book  is  devoted  to  a discussion  of  fundamental 
facts,  while  in  the  second  half  the  practical  application  of  bacteriology 
to  agriculture  has  been  fully  considered.  Many  problems  of  great 
importance  to  the  farmer  are  treated  in  the  chapters  on  Dairy  and  Soil 
Bacteriology,  but  their  accurate  understanding  is  not  assured  unless 
the  preceding  chapters  have  also  been  studied. 

The  book  is  neither  a laboratory  manual  nor  a reference  book,  but 
the  quotations  given  in  the  text  and  in  footnotes  will  direct  the  reader 
to  such  literature  if  this  is  desired. 

We  are  indebted  to  Professor  E.  G.  Hastings  for  valuable  criticisms. 
With  a few  exceptions  the  illustrations  are  originals,  mostly  made  in 
the  senior  author’s  laboratory  at  Leipzig.  About  twelve  new  ones 
have  been  prepared  by  F.  L.  Goll  of  the  U.  S.  Department  of  Agri- 
culture. 

Washington,  D.  C.  F.  LoHNIS. 

Madison,  Wis.  g g Fred. 

December,  1922. 

v 


281124 


Digitized  by  the  Internet  Archive 
in  2016  with  funding  from 
Duke  University  Libraries 


https://archive.org/details/textbookofagricu01lohn 


CONTENTS 


PAGE 

Introduction 1 

Relation  of  bacteriology  to  agriculture — History  of  bacteriology — ■ 
Literature. 

PART  I 

GENERAL  MORPHOLOGY  AND  PHYSIOLOGY  OF  BACTERIA 
AND  RELATED  MICROORGANISMS 

CHAPTER 

I.  Morphology  of  Bacteria  and  Related  Microorganisms  ....  15 

Form  and  size  of  cells — Variability  of  the  cell  form;  involution  forms — 
Monomorphism  and  pleomorphism — Cell  compounds;  branched  growth — 
Structure  of  cells — Flagellation. 

II.  Development  of  Bacteria  and  Related  Microorganisms  ...  25 

Multiplication  of  cells — Formation  of  colonies — Conjunction,  conjugation, 
copulation — Reproductive  organs  and  resting  cells — Autolysis  and  sym- 
plastic  stage. 

III.  Classification  of  Bacteria,  Fungi,  and  Protozoa 34 

Artificial  and  natural  classification — Nomenclature — Various  systems  of 
bacteria,  lower  fungi,  and  protozoa. 

IV.  Relations  of  Microorganisms  to  Their  Environment  ....  39 

1.  Bacterial  Nutrition 39 

Chemical  composition  of  cells — Nutrients — Stimulants — Alkaline  and 
acid  reactions. 

2.  Physical  Factors 44 

Moisture — Air — Temperature — Light — Various  other  factors. 

3.  Symbiosis  and  Antagonism 54 

4-  Resistance  of  Resting  Forms 58 

5.  Distribution  of  Microorganisms  in  Nature 60 

Occurrence  in  soil,  air,  water,  milk,  and  manures — Adaptation  to  the 
environment. 

V.  Counting,  Isolating,  Cultivating,  and  Testing  Bacteria  and  Related 

Microorganisms 67 

Counting  bacteria,  fungi,  and  protozoa — Plate  cultures — Single-cell 
cultures — Testing  pure  cultures. 

VI.  Sterilization,  Pasteurization,  Antisepsis,  and  Asepsis  ...  78 

Effect  of  various  methods — Physical  treatment — Chemical  treatment — 
Combined  treatments. 

vii 


281124 


CONTENTS 


viii 

CHAPTER  PAGE 

VII.  Activities  of  Bacteria  and  Related  Microorganisms  ....  86 

Efficiency  and  virulence — Physical  and  chemical  actions. 

1.  Production  of  Color,  of  Light,  and  of  Heat 88 

2.  Transformation  of  Organic  Substances 93 

Putrefaction,  decay,  fermentation — Nitrogen-carbon  ratio — Enzymes. 

8.  The  Cycle  of  Nitrogen 95 

Destruction  of  organic  nitrogenous  compounds — Ammonification — Nitri- 
fication— Nitrate  reduction — Assimilation  of  amino,  ammonia,  and 
nitrate  nitrogen — Liberation  of  nitrogen — Fixation  of  nitrogen. 

4-  The  Cycle  of  Carbon,  Oxygen,  and  Hydrogen 117 

Metabolism  of  carbohydrates,  alcohols,  and  organic  acids — Formation 
and  destruction  of  humus — Formation  and  assimilation  of  carbon  dioxide 
— Formation  and  metabolism  of  carbon  monoxide,  methane,  and  hydrogen. 

5.  Transformation  of  Mineral  Substances 130 

Metabolism  of  phosphorus  compounds — Bacterial  action  upon  carbonates 
and  silicates — Sulfur  bacteria — Iron  bacteria. 

6.  Pathogenic  Action  of  Microorganisms 138 

Virulence  and  infection — Immunity  and  immunization — Vaccination, 
serum  treatment,  and  chemotherapy. 

PART  II 

DAIRY  AND  SOIL  BACTERIOLOGY 

VIII.  Bacteria  and  Related  Microorganisms  in  Foodstuffs  . . . 149 

Germ  content — Participation  of  bacteria  and  fungi  in  the  making  of  hay, 
silage,  and  sauerkraut — Spoilage  of  fodder — Activities  of  bacteria  in  the 
digestive  tract. 

IX.  Bacteria  and  Related  Microorganisms  in  Milk 161 

1.  Germ  Content  of  Milk 161 

Modes  of  contamination— Reduction  and  increase  in  numbers — Different 
qualities  of  milk — Biological  milk  tests. 

2.  Activities  of  Bacteria  in  Milk 176 

Normal  and  abnormal  alterations  of  milk — Formation  of  acids,  alcohol, 
and  gas — Decomposition  of  casein  and  fat — Changes  in  taste,  flavor, 
color,  and  viscosity. 

8.  Milk  Pasteurization — Fermented  Milks 184 

X.  Bacteria  and  Related  Microorganisms  in  Butter  ....  188 

1 . Germ  Content  of  Butter 188 

Origin  of  butter  organisms — Changes  in  germ  content — Types  of  butter 
organisms. 

2.  Bacterial  Action  and  Quality  of  Butter 191 

Influence  upon  taste  and  flavor — Changes  in  storage  butter — Rancidity 
Other  abnormal  alterations. 

8.  Cream  Pasteurization — Use  of  Starters 


195 


CONTENTS 


IX 


CHAPTER  PAGE 

XI.  Bacteria  and  Related  Microorganisms  in  Cheese  ....  197 

1.  Germ  Content  of  Cheese 197 

Origin  of  cheese  organisms — Frequency  and  species  of  microorganisms  in 
cheese. 

2.  Bacterial  Activities  and  the  Ripening  of  Cheese 201 

Enzymatic  and  bacterial  activities — Normal  and  abnormal  alterations 

in  ripening  cheese. 

3.  Means  of  Regulating  the  Activity  of  Microorganisms  in  Cheese  . . . 210 

Influence  of  technique— Use  of  pasteurized  milk  and  of  starters 

XII.  Sewage  Disposal 215 

Various  methods — Septic  tanks — Trickling  filters — Activated  sludge — 
Chemical  treatment. 

XIII.  Bacteria  and  Related  Microorganisms  in  Barnyard  Manures  . .221 

1.  Germ  Content  of  Barnyard  Manures 222 

Frequency  and  groups  of  microorganisms. 

2.  Bacterial  Activities  in  Barnyard  Manures 224 

The  rotting  of  manure — Decomposition  of  carbonaceous  and  nitrogenous 
compounds — Liberation  and  fixation  of  nitrogen. 

3.  Prevention  of  Losses  of  Plant  Food  from  Barnyard  Manure  ....  234 
Mechanical,  chemical,  and  biological  methods. 

XIV.  Bacteria  and  Related  Microorganisms  in  Soils 237 

1 . Germ  Content  of  Soils 237 

Quantity  and  quality  of  soil  organisms — Their  relation  to  the  produc- 
tivity of  soils — Boil  sickness — Biological  soil  tests. 

2.  Bacterial  Activities  in  Soils 244 

Carbon  metabolism — Humus,  tilth,  and  productivity  of  the  soil — Nitrogen 
metabolism — Losses  and  gains  in  nitrogen. 

3.  Means  of  Regulating  the  Activities  of  Microorganisms  in  the  Soil  . . 262 

Influence  of  soil  management  (tillage,  irrigation,  manuring,  liming,  crop- 
ping, fallowing) — Soil  disinfection — Inoculation  of  soils  and  seeds. 

Index 273 


TEXTBOOK 


OF 

AGRICULTURAL  BACTERIOLOGY 


INTRODUCTION 

During  the  last  decades  bacteriology  has  become  of  great  importance 
to  agriculture,  because  bacteria  may  act  in  many  ways,  both  useful 
and  harmful  to  the  farmer.  Molds,  yeasts,  lower  algae,  and  protozoa 
frequently  participate  in  such  processes.  A general  term  applicable 
to  all  these  minute  living  beings  is  “microorganisms”  or  “microbes.”  1 
Accordingly,  “microbiology”  is  another  name  for  this  branch  of  science, 
and  this  term  is  really  more  nearly  accurate  than  the  commonly 
used  denomination  “bacteriology.”  However,  the  latter  expression  is 
fairly  well  established  not  only  because  it  has  been  in  vogue  for  several 
decades,  but  also  on  account  of  the  fact  that  in  most  of  the  processes 
concerned,  bacteria  usually  exert  the  greatest  influence. 

Aims  and  Scope  of  Bacteriology. — Bacteria  and  related  microor- 
ganisms may  participate  in  the  following  processes: 

1.  Human  diseases 

2.  Animal  diseases 

3.  Plant  diseases 

4.  Normal  and  abnormal  alterations  of  foodstuffs,  of  milk,  butter  and  cheese. 

5.  Numerous  biochemical  processes  taking  place  in  sewage,  in  manure,  and  in  soil. 

Bacteria  have  gained  their  widest,  though  unfavorable,  reputation 
as  causative  agents  of  human  and  animal  diseases.  Many  people  con- 
sider the  term  bacteria  as  practically  synonymous  with  infection,  disease, 
and  death.  This  belief,  however,  is  no  less  incorrect  than  it  would  be 
to  assume  that  all  green  plants  are  dangerous,  because  a few  of  them 
are  poisonous.  Nevertheless,  general  interest  was  first  attracted  by 
medical  bacteriology,  and  the  discovery  of  bacteria  as  causative  agents 
of  human  and  animal  contagious  diseases  made  the  term  bacteriology 

1 Derived  from  the  Greek  words  nwpbs  (mikros)  small,  and  /Sios  (bios)  life. 


2 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


familiar  to  civilized  mankind.  Bacteriology  soon  became  an  important 
branch  of  both  human  and  veterinary  medicine  and  has  attained  an 
enormous  development  within  less  than  half  a century. 

Plant  diseases  also  have  been  thoroughly  studied  for  a considerable 
length  of  time.  In  most  cases  fungi  were  discovered  as  causative  agents ; 
but  more  recently  some  important  plant  diseases  were  found  to  be 
caused  by  bacteria.  Their  study,  therefore,  has  become  a part  of  plant 
pathology. 

Agricultural  Bacteriology. — The  two  main  objects  of  agricultural 
bacteriology  are:  (a)  the  study  of  bacteria  and  other  microorganisms  in 
their  relation  to  foodstuffs,  milk,  and  dairy  products,  usually  called 
dairy  bacteriology ; and  (b)  the  study  of  the  bacterial  processes  in 
manure  and  in  soil,  usually  termed  soil  bacteriology. 

The  enormous  amount  of  knowledge  accumulated  with  regard  to 
the  causative  agents  of  human,  animal,  and  plant  diseases,  as  well  as 
the  multitude  of  problems  awaiting  further  investigation,  have  made 
it  unavoidable  that  these  subjects  were  again  subdivided  among  special- 
ists. The  same  tendency  of  specialization  is  also  noticeable  in  agricul- 
tural bacteriology.  Some  laboratories  are  reserved  for  dairy  bac- 
teriology, others  for  soil  bacteriology,  a few  for  still  more  specialized 
work.  The  thoroughness  required  for  successful  research  work  neces- 
sitates far-reaching  specialization,  but  in  order  to  obtain  a broad  and 
well  balanced  knowledge  of  the  whole  field  of  agricultural  bacteriology 
attention  should  not  be  centered  too  much  upon  certain  special  prob- 
lems, although  they  may  at  once  attract  high  interest  on  account  of 
their  great  practical  importance.  Sound  knowledge  of  the  fundamental 
facts  is  the  basis  upon  which  all  specialization  must  rest,  and  the 
agriculturist  who  possesses  such  knowledge  will  not  find  it  very  difficult 
to  gain  a correct  understanding  of  new  findings  in  agricultural  bac- 
teriology, and  to  make  proper  practical  application  of  them,  if  this  is 
feasible. 

Cycle  of  Matter. — To  what  extent  agriculture  and  even  the  con- 
tinuity of  Life  itself  depend  on  the  incessant  and  energetic  activity 
of  bacteria  and  related  microorganisms  can  easily  be  demonstrated. 
Figure  1 shows  in  a schematic  manner  how  the  eternal  cycle  of  matter 
is  used  and  regulated  by  agriculture  and  industry  for  the  benefit  of 
mankind.  The  mineral  constituents  of  the  soil  help  to  build  up  plant 
products.  These  may  be  either  directly  used  as  human  food,  or  they 
may  be  converted  into  animal  products,  such  as  meat  and  milk,  or  into 
industrial  products,  such  as  linen  and  cotton  garments,  vegetable  oils, 
etc.  Animal  and  industrial  products  again  may  be  either  directly  used, 
or  they  may  undergo  another  transformation  before  they  serve  our 


INTRODUCTION 


3 


purposes;  for  instance,  wool  is  transformed  into  cloth,  industrial 
residues  like  oil  cakes  are  used  as  fodder,  etc.  But  all  these  con- 
structive processes  would  soon  come  to  an  end  if  there  were  not  a 
complete  cycle  of  matter,  that  is,  if  the  material  used  by  plants, 
animals,  and  men,  would  not  ultimately  and  regularly  return  to  its 
origin.  All  organic  residues  must  again  be  mineralized,  otherwise  the 
earth  would  long  since  have  been  littered  with  corpses,  and  all  life 
would  have  become  extinct.  It  is  true  that  by  the  respiration  of  living 


plants,  animals,  and  men  considerable  quantities  of  organic  substances 
are  being  constantly  broken  up  into  carbon  dioxide  and  water,  but 
this  fact  does  not  materially  change  the  general  necessity  of  a per- 
manent equilibrium  between  constructive  and  destructive  processes, 
between  Life  and  Death. 

Work  of  Higher  and  of  Lower  Organisms. — That  the  constructive 
part  of  the  cycle  of  matter  is  closely  connected  with  the  life  of  organ- 
isms was  always  self-evident  to  thinking  mankind.  On  the  other  hand, 
only  during  the  last  decades  was  it  discovered  that  nearly  every  step 


Food 

L^and  Clothing 


Organic  Constituents 


of  Soil 


Fig.  1. — Cycle  of  matter. 


4 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


in  the  retrograde  transformation  of  organic  substances  represents  the 
work  of  minute  organisms,  which  can  not  be  seen  except  by  the  use 
of  most  powerful  optical  instruments.  Occasionally  purely  physical 
and  chemical  processes  participate  in  these  destructive,  as  well  as  in 
the  constructive,  changes  of  matter.  Nevertheless,  the  dominating  and 
directing  influence  of  living  organisms  is  now  equally  beyond  doubt 
in  both  cases.  The  microorganisms  in  milk,  in  butter,  in  cheese,  in 
manure,  and  in  soil  are  just  as  important  to  the  farmer,  although  he 
may  never  see  them,  as  are  the  milk  cows  hi  his  stable  and  the  growing 
crops  in  his  fields. 

The  task  of  the  medical  bacteriologist  usually  centers  upon  the 
problem  of  becoming  acquainted  with  the  disease-producing  germ,  and 
to  find  out  how  it  can  be  successfully  fought  and  eliminated.  The 
farmer,  however,  should  know  under  what  conditions  he  will  be  able 
to  secure  the  most  favorable  results  from  the  cooperation  of  the  useful 
bacteria,  and  how  to  avoid  the  detrimental  effects  of  the  activities 
of  harmful  microorganisms.  It  was  an  old  belief  among  practical 
agriculturists  that  barnyard  manure  adds  “life”  to  the  soil,  that  the 
surface  soil  is  more  active  than  the  “inert”  subsoil,  that  the  “ripen- 
ing” of  cream  and  cheese  depends  to  a great  extent  on  the  use  of 
well  “working”  starters.  These  and  similar  expressions  indicate 
clearly  how  by  practical  experience  a fairly  correct  insight  was  gained 
long  before  exact  bacteriological  investigations  had  become  possible. 

Environmental  Conditions. — The  excellent  results  obtained  by  care- 
ful selection  and  breeding  of  the  cultivated  plants  and  domesticated 
animals  led  many  to  the  belief  that  it  should  be  the  foremost  task 
of  the  agricultural  bacteriologist  to  select  and  to  cultivate  the  most 
efficient  strains  of  useful  bacteria  in  order  to  make  them  available  for 
the  practical  agriculturist.  However,  only  in  a few  cases  can  such 
direct  results  be  expected,  as  for  instance  in  the  use  of  selected  bacterial 
cultures  for  the  preparation  of  starters  in  the  dairy,  or  for  the  inocula- 
tion of  leguminous  seeds.  In  all  other  cases  the  conditions  under 
which  these  useful  microorganisms  live  and  work  must  first  be  in- 
vestigated very  thoroughly.  Even  the  most  active  bacteria  can  not  dis- 
play their  ability  under  unfavorable  conditions,  just  as  the  best  milk 
cow  cannot  show  a high  productivity  when  improperly  kept  and  fed, 
nor  will  the  best  seed  ever  produce  heavy  crops  on  a badly  tilled,  weedy 
soil. 

To  secure  a complete  and  detailed  knowledge  of  these  environmental 
conditions  is  by  no  means  an  easy,  though  a very  important  task  of 
agricultural  bacteriology.  At  the  present  much  remains  to  be  done 
in  this  direction,  and  frequently  one  must  be  satisfied  if  at  least  the 


INTRODUCTION 


5 


general  principles  have  been  worked  out  which  are  governing  bacterial 
life  in  the  different  phases  of  the  transformation  of  matter.  Year  by 
year  more  details  will  be  discovered;  but  a clear  impartial  conception 
of  their  accuracy  and  importance  will  always  be  dependent  on  a sound 
knowledge  of  the  underlying  general  principles.  Therefore  these  will 
have  to  be  considered  before  the  various  problems  of  dairy  and  soil 
bacteriology  can  be  approached  intelligently.  A short  historical  survey 
of  the  development  of  bacteriology  will  be  given  first. 

Earliest  Bacteriological  Hypotheses. — A more  or  less  indistinct  feel- 
ing that  many  of  the  processes  now  known  to  be  caused  by  bacteria 
were  an  expression  of  some  invisible  life  may  be  traced  back  through 
many  centuries.  About  two  thousand  years  ago  an  agricultural  text- 
book was  written  by  Marcus  Terentius  Varro,  wherein  it  is  emphasized 
that  farm  buildings  never  should  be  erected  on  swampy  ground.  As  one 
of  the  reasons  for  this  advice  the  author  states  that  in  such  land  “certain 
minute  invisible  animals  develop  which,  transferred  by  the  air,  may 
enter  the  body  through  mouth  or  nose,  and  may  cause  serious  diseases.” 
In  its  original  form  this  interesting  piece  of  antique  bacteriology  reads 
as  follows : 1 

Advertendum  etiam,  si  qua  erunt  loca  palus'tria,  . . . quod  in  iis  crescunt 
animalia  quaedam  minuta,  quae  non  possunt  oculi  c-onsequi,  et  per  aera  intus 
in  corpus  per  os  ac  nares  perveniunt  atqne  effieiunt  difficiles  morbos. 

It  must  be  left  in  doubt  whether  Varro  himself  was  the  first  to  con- 
ceive this  remarkably  accurate  idea,  or  whether  he  merely  copied  it  from 
an  older  unnamed  source.  Palladius,  author  of  another  book  “On  Agri- 
culture,” wrote  again  about  400  years  later:2 

Palus  omui  modo  vitanda  est,  . . . propter  pestilentia  vel  animalia  inimica, 
quae  generat. 

(Swamps  must  be  avoided  because  of  the  plagne  or  the  dangerous  animals 
which  develop  therein.) 

The  beneficial  effect  of  the  nitrogen  fixing  bacteria  now  known  to  be 
active  in  the  root  nodules  of  leguminous  plants,  was  also  fairly  well  known 
among  the  agricultural  writers  of  ancient  Rome.  Planting  of  lupine, 
vetch,  bean,  etc.,  was  declared  to  enrich  the  soil  and  to  act  like  an  applica- 
tion of  barnyard  manure.  In  Columella’s  book  “De  re  rustica,”  written 
in  the  first  century  of  the  Christian  era,  we  find  lupine,  alfalfa,  vetch, 
bean,  lentil,  chick-pea,  and  pea  enumerated  as  plants  which  either  enrich 

1 Varronis  de  re  rustica,  Lib.  I,  cap.  XII,  printed  in  1536  by  Joannes  Gymnicus  in 
Cologne,  together  with  contributions  “De  re  rustica”  by  Cato,  Palladius,  and  Columella. 

2 Palladii  de  re  rustica, — Lib.  I,  tit.  VII. 


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the  soil  or  at  least  preserve  its  fertility,  while  all  others  are  said  to 
exhaust  the  fields.1 

Earliest  Bacteriological  Observations. — In  numerous  mediaeval  pub- 
lications the  doctrine  of  the  “contagium  animatum”  (the  living  con- 
tagion) was  treated  again  and  again;  certain  “animalcula”  were  sup- 
posed to  be  responsible  for  various  infectious  diseases.  The  first 
investigators  who  actually  succeeded  in  seeing  bacteria  were  probably 
the  two  Dutchmen  Anthony  van  Leeuwenhoek  and  Christian  Huygens. 
Leeuwenhoek  himself  made  the  lenses  for  his  manifold  studies,  upon 
which  he  reported  in  numerous  letters  to  the  Royal  Society  of  London. 
A complete  collection  of  these  communications  was  printed  in  1695  at 
Delft,  where  he  resided,  under  the  title  “Arcana  Naturae  Detecta” 


Fig.  2. — Drawings  of  bacteria  made  by  A.  van  Leeuwenhoek  in  1683  (Figs.  A-G  on 
left  side)  and  in  1693  (Figs.  A-D  on  right  side),  reproduced  in  “Arcana  Naturae 
Detecta,”  1695,  pp.  42  and  335. 


(Nature’s  Secrets  Unveiled).  The  first  reference  to  bacterial  life  is  to 
be  found  in  a letter  dated  October  9,  1676,  and  two  very  interesting  sets 
of  drawings  of  bacteria  were  presented  in  two  other  letters,  written 
in  1683  and  in  1692;  both  are  reproduced  in  Fig.  2.  A few  years  older 
than  these  are  some  drawings  made  by  Chr.  Huygens  in  a manuscript 
dating  from  1678. 2 

Leeuwenhoek  used  for  his  sketches  material  taken  from  his  teeth, 
which,  as  he  emphasizes,  were  perfectly  clean  and  healthy.  Some  of  the 
bacteria  were  found  to  be  actively  motile,  or  as  the  Dutch  author  says 
“very  gayly  moving”  under  his  lenses  (indicated  by  the  curved  dotted 
line  C-D  in  Fig.  2).  In  rainwater  and  in  watery  infusions  of  various 

1 Columella,  Lib.  II,  cap.  X,  XI,  and  XIV. 

2 Beijerinck,  Jaarboek  der  K.  Akad.  Amsterdam,  1913. 


INTRODUCTION 


7 


organic  substances  he  saw  similar  organisms ; but  he  did  not  enter  into 
any  hypotheses  or  investigations  concerning  the  role  these  minute 
“animals”  were  possibly  playing  in  nature. 

Earliest  Bacteriological  Experiments. — An  abstract  of  Leeuwen- 
hoek’s letter  of  1683  was  published  ten  years  later  in  the  Philosophical 
Transactions  of  the  Royal  Society  of  London  (Vol.  XVII,  1693).  A 
few  weeks  afterwards  Sir  Edmond  King,  a member  of  this  society,  con- 
firmed the  correctness  of  the  Dutch  author’s  findings,1  and  also  pointed 
out  some  important  physiological  facts  which,  however,  were  soon  for- 
gotten. To  ascertain  exactly  whether  these  minute  corpuscles  were  really 
living  beings,  he  added  with  a needle  small  amounts  of  sulfuric  acid, 
ink,  salt,  sugar,  or  fresh  blood  to  the  droplets  containing  the  bacteria 
under  his  microscope.  Sulfuric  acid  and  fresh  blood  proved  to  be  most 
injurious;  they  quickly  killed  the  bacteria,  while  the  other  substances 


Fig.  3. — Drawings  made  by  Sir  Edm.  King,  Philos.  Transact.  Roy.  Soc.  (London) 

Vol.  XVII,  1693,  No.  203. 

merely  caused  a temporary  shrinking  or  swelling  of  the  cells.  By  adding 
fresh  water  the  original  cell  form  could  be  reestablished,  provided  that 
the  alteration  had  not  gone  too  far  and  had  not  yet  caused  the  death 
of  the  organism.  This  report  shows  that  three  facts,  generally  considered 
to  be  quite  recent  discoveries,  i.e.,  the  bactericidal  action  of  blood,  the 
plasmolysis  and  plasmoptysis  of  bacterial  cells  (to  be  discussed  in 
Chapters  I and  VII,  6)  are  clearly  described  in  this  early,  but  long  for- 
gotten paper.  Some  drawings  made  by  King  are  reproduced  in  Fig.  3 ; 
they  are  unquestionably  inferior  to  those  of  Leeuwenhoek. 

Earliest  Bacteriological  Classification. — During  the  eighteenth  cen- 
tury many  more  or  less  ingenious  speculations  were  contributed  by 
various  authors,  but  only  one  real  advance  in  bacteriology  was  to  be 
recorded.  It  is  represented  by  the  appearance  of  a beautifully  illustrated 
book  on  “Infusoria,”  written  by  the  Danish  investigator  O.  F.  Muller ,2 


1 King,  Phil.  Trans.  Roy.  Soc.,  vol.  XVII,  1693,  pp.  861-865. 

2 “Animalcula  infusoria  fluviatilia  et  marina.”  Hauniae,  1786. 


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Several  of  the  generic  names  introduced  by  him  (Monas,  Vibrio,  Proteus) 
are  retained  up  to  the  present  time. 

Earliest  Practical  Application  of  Bacteriology. — Early  in  the  nine- 
teenth century  the  first  practical  results  in  bacteriology  were  secured: 
The  Frenchman  Appert  discovered  and  taught  the  principles  of  success- 
fully preserving  animal  and  vegetable  foods.1  A better  knowledge  of 
the  various  possibilities  of  thorough  disinfection  was  also  gained.  That 
in  some  respects  our  forefathers  had  indeed  fairly  accurate  ideas  is  to 
be  seen,  for  instance,  from  what  was  known  at  that  time  about  the  cause 
and  remedy  of  the  blue  discoloration  of  milk  kept  in  cellars.  Some  kind 
of  fungus  was  believed  to  settle  on  the  surface  of  the  milk;  fumigation 
by  burning  sulfur  and  treatment  of  the  vessels  with  hydrochloric  acid 
were  strongly  recommended.2 

In  1837  Th.  Schwann 3 stated  definitely  that  all  fermentative  and 


abed. 

Fig.  4.— Drawings  of  bacteria  published  by  Pasteur  in  1864  (Compt.  rend,  tome  58, 
p.  142).  (a)  Urea  bacteria.  (5)  Lactic  acid  bacteria  and  yeasts,  (c)  and  ( d ) Butyric 


acid  bacteria. 

putrefactive  processes  are  caused  by  living  organisms  (“infusoria”  and 
fungi).  Two  years  later  it  was  emphasized  by  Donne  4 that  the  altera- 
tions in  milk  should  be  investigated  not  only  by  chemical  methods,  but 
under  the  microscope  too.  Soon  after  C.  J.  Fuchs  5 succeeded  in  clearing 
up  the  bacterial  causes  of  the  souring  as  well  as  of  several  abnormal 
changes  of  milk. 

Louis  Pasteur  and  His  Contemporaries. — The  real  foundation  of 
modern  microbiology,  however,  was  laid  by  the  famous  French  chemist, 
Louis  Pasteur.  Since  1857  he  published  in  the  “Comptes  rendus  de 
l’Academie  des  sciences  a Paris”  numerous  papers  on  fermentation, 
formation  of  lactic  acid  and  butyric  acid,  transformation  of  urea  to 
ammonia,  etc.  It  is  true  that  his  first  object  was  to  disprove  the  old 

1 “L’art  de  conserver  toutes  les  substances  animales  et  vdge  tales,”  1810. 

2 A.  Thaer,  “Grundsatze  der  rationellen  Landwirtschaft,”  Bd.  4,  6.  Hauptstuck,  § 54. 

3 Annalen  der  Physik  und  Chemie,  2.  Folge,  Bd.  41,  p.  184. 

4 Compt.  rend.  Acad.  Paris,  tome  9,  pp.  367,  800. 

6 Magazin  fur  die  gesamte  Thierheilkunde,  Bd.  7,  1841,  pp.  150,  174,  180-194. 


INTRODUCTION 


9 


hypothesis  of  spontaneous  generation,  which  was  revived  once  more  at 
that  time,  but  soon  his  studies  turned  to  more  important  and  more 
practical  problems,  and  they  stimulated  effectively  similar  research  work, 
especially  in  France  and  in  England. 

An  immediate  practical  application  of  Pasteur’s  discoveries  to  agri- 
cultural problems  was  advocated  as  early  as  1862  in  a booklet  written 
by  a German  farmer  named  W.  Iiette.1  It  was  emphasized  therein  that 
in  addition  to  the  chemical  points  of  view,  as  taught  at  that  time  by 
J.  Liebig  and  his  disciples,  the  biological  aspects  also  should  be  con- 
sidered, especially  with  regard  to  the  effect  of  stable  manure  and 
green  manures  upon  the  tilth  of  the  soil.  Decades  passed,  however, 
before  this  advice  was  heeded. 

Development  of  Dairy  Bacteriology. — More  rapid  progress  was 
made  in  the  microbiology  of  milk  and  dairy  products.  For  ex- 
ample, von  Hessling2  wrote  in  1866  quite  positively  that  as  the  various 
fermentations  in  milk  so  also  the  ripening  of  cheese  is  caused  by  lower 
fungi ; and  in  a book  entitled  ‘ ‘ Etudes  sur  la  fabrication  de  f romage,  ’ ’ 
published  in  1867  by  L.  II.  de  Martin,  the  differences  among  the  various 
kinds  of  cheese  were  explained  as  the  results  of  the  activity  of  different 
species  or  of  various  varieties  of  microorganisms.  The  intelligent 
use  of  starters  for  cream  and  cheese  ripening  was  explained  and  recom- 
mended in  several  books  of  that  time.  More  detailed  information  con- 
cerning the  bacteria  connected  with  the  ripening  of  cheese  was  sought 
and  secured  by  E.  Duclaux  in  France,3  and  by  Manetti  and  Musso  in 
Italy,4  while  the  famous  British  surgeon  John  Lister  5 worked  on  the 
problem  of  excluding  all  bacteria  from  the  milk  by  observing  the  greatest 
cleanliness  in  every  respect.  Soon  after,  in  1884,  another  Englishman, 
0.  Ernest  Pohl,  made  use  of  these  investigations  and  was  indeed  able 
to  produce  on  his  farm  milk  of  very  low  germ  content 6 by  anticipating 
those  methods  which  are  now  recommended  by  the  American  Medical 
Milk  Commissions  for  the  production  of  certified  milk. 

Development  of  General  and  of  Soil  Bacteriology. — Very  thorough 
botanical  investigations  upon  the  morphology  and  physiology  of  the 
bacteria  were  started  in  1872  by  Ferdinand  Cohn  at  the  University  of 
Breslau,7  and  it  was  in  this  laboratory  that  Robert  Koch  developed 

1 “Die  Ferments tionstheorie  gegeniiber  der  Humus-,  Mineral-  und  Stickstoff- 
theorie.”  Berlin,  1862. 

2 Virchow’s  Archiv  f.  pathol.  Anatomie,  Bd.  35,  p.  561. 

3 Ann.  agronomiques,  1879,  Ann.  de  I'lnstitut  national  agromique,  1879-80. 

kLandw.  Versuchsstationen,  Bd.  21,  1878,  p.  224. 

6 Quarterly  Jour,  of  Microscopical  Science,  New  Series,  vol.  18,  1878,  p.  179. 

6 Helbig,  Pharmazeutische  Zentralhalle,  Bd.  51,  1910,  p.  1051. 

7 Beitrdge  zur  Biologie  der  Pflanzen,  1872-1876. 


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his  ingenious  methods  which  became  the  basis  of  modern  bacteriology. 
In  1876  his  classical  work  on  the  anthrax  bacillus  was  published  1 ; a few 
years  later  he  also  completed  the  first  extensive  work  on  the  bacterial 
content  of  the  soil.2  Prior  to  these  studies,  however,  were  the  very 
thorough  investigations  on  nitrification  in  soil,  made  by  the  French 
chemists  TTi.  Schlosing  and  A.  Muntz, 3 which  were  later  confirmed  and 
extended  at  the  Bothamsted  laboratory  in  England  by  R.  Warington ,4 
who  also  worked  on  denitrification  and  other  important  biological  proc- 
esses taking  place  in  the  soil. 

Discoveries  on  Nitrogen  Fixation. — At  Bothamsted,  as  at  other 
places,  interesting  data  had  been  collected  with  regard  to  the  fixation  of 
nitrogen  by  leguminous  plants,  but  it  remained  for  the  German  chemists 
Hellriegel  and  Wilfarth  5 to  secure  complete  and  final  proof  that  again 
bacteria,  living  in  the  nodules  peculiar  to  the  roots  of  these  plants,  are 
directly  connected  with  this  process.  In  1888  the  Dutch  bacteriologist 
M.  W.  Beijerinck  succeeded  in  obtaining  pure  cultures  of  these  organ- 
isms, and  soon  after  he  was  able  to  show  that  they  indeed  are  the  causes 
of  root  nodules  and  nitrogen  fixation.0  Until  1921,  Beijerinck  continued 
to  work  at  Delft  (the  same  ancient  town  where  more  than  200  years 
earlier  Leeuwenhoek  had  made  his  first  contributions  to  bacteriology)? 
and  many  discoveries  of  great  importance  to  agricultural  bacteriology 
have  originated  in  his  laboratory.  Best  known  among  them  is  perhaps 
his  work  on  Azotobacter,  the  most  vigorous  of  the  numerous  bacteria 
capable  of  enriching  the  soil  by  the  fixation  of  atmospheric  nitrogen.7 
Other  nitrogen  assimilating  bacteria  had  been  cultivated  before  by  Mar- 
cellin  Berthelot 8 in  France,  and  by  S.  Winogradsky  9 in  Bussia.  The 
latter  also  succeeded  for  the  first  time  in  the  difficult  task  of  growing  pure 
cultures  of  the  nitrifying  organisms.10 

Present  Status  of  Medical  and  of  Agricultural  Bacteriology. — After 
Bobei't  Koch  and  his  disciples  had  solved,  in  the  early  eighties  of  the 

1 In  Cohn’s  Beitragen  zur  Biologie  der  Pflanzen,  Bd.  2,  Heft  2,  p.  277. 

2 Mitteilungen  aus  dem  Kaiserl.  Gesundheits-Amte,  Bd.  1,  1881,  p.  34. 

3 Compt  rend.  Acad.  Paris,  tome  77,  1873,  tome  84  and  85,  1877,  tome  86,  1878, 
and  tome  89,  1879. 

4 U.  S.  Dept.  Agr.,  Exp.  Sla.  Bull.  8,  1892. 

6 Landw.  Versuchsstationen,  Bd.  33,  1886,  Bd.  34,  1887,  and  “Untersuchungen 
uber  die  Stickstoffnahrung  der  Gramineen  und  Leguminosen,”  Beilageheft  z.  Zeitschr.  d. 
Vereinsf.  Rubenzuckerindustrie,  Nov.,  1888. 

6 Botanische  Zeitung,  Bd.  46,  1888,  Bd.  48,  1890. 

7 Centralbl.  f.  Bakt.,  II.  Abt.,  Bd.  7,  1901,  p.  567. 

8 Compt.  rend.  Acad.  Paris,  tome  116,  1893,  p.  843. 

9 Compt.  rend.  Acad.  Paris,  tome  116,  1893,  p.  1385. 

10  Annales  de  Vlnstitut  Pasteur,  tome  5,  1891;  Archives  des  sciences  biologiques,  St. 
Petersbourg,  tome  1,  1892. 


INTRODUCTION 


11 


last  century,  the  old  questions  concerning  the  causative  agents  of  such 
dreaded  diseases  as  cholera,  tuberculosis,  typhoid,  etc.,  the  medical  branch 
of  bacteriology  spread  rapidly  to  all  civilized  nations,  and  laboratories 
for  medical  bacteriology  were  established  everywhere.  With  agricultural 
bacteriology,  progress  was  slower.  In  Germany,  the  one-sided  chemical 
point  of  view,  as  established  by  J.  Liebig,  remained  predominant.  In 
France,  in  England,  and  in  other  European  countries  the  investigators 
were  not  numerous  enough  to  exert  a marked  influence.  So  it  became 
the  opportunity  of  America  to  offer  a promising  field  to  bacteriological 
investigators.  In  addition  to  numerous  research  laboratories  for 
medical  bacteriology,  the  agricultural  branch  of  this  science  is  equally 
well  represented  at  the  American  agricultural  experiment  stations. 
During  the  last  decades  much  progress  has  been  made  in  this  country, 
and  at  present  about  1000  workers  are  united  in  the  ‘ ‘ Society  of  American 
Bacteriologists.”  If  the  opportunities  offered  are  adequately  used,  many 
valuable  results  may  be  expected,  because  many  problems  are  awaiting 
thorough  investigation. 

Literature. — A classified  list  of  books  and  periodicals  devoted 
wholly  or  in  part  to  agricultural  bacteriology  is  given  below.  The 
large  reference  books,  mentioned  under  C,  will  furnish  information 
on  special  subjects. 

A.  Textbooks  on  General  Bacteriology 

W.  Benecke,  Bau  und  Leben  der  Bakterien,  1912. 

E.  O.  Jordan,  Textbook  of  General  Bacteriology,  1922. 

W.  Kruse,  Allgemeine  Mikrobiologie,  1910. 

E.  Mace,  Traite  de  Microbiologie,  1912-1913. 

Ch.  Marshall,  Microbiology,  1921. 

B.  Textbooks  on  Agricultural  Bacteriology 

H.  W.  Conn,  Agricultural  Bacteriology,  1918. 

J.  E.  Greaves,  Agricultural  Bacteriology,  1922. 

E.  Kayser,  Microbiologie  agricole,  1921. 

S.  Orla-Jensen,  Dairy  Bacteriology,  1921. 

E.  Pantanelli,  Prinzipali  Fermentazioni  dei  Prodotti  Agrari,  1912. 

J.  Percival,  Agricultural  Bacteriology,  1920. 

H.  L.  Russell  and  E.  G.  Hastings,  Agricultural  Bacteriology,  1921. 

C.  Reference  Books  on  General  and  Agricultural  Bacteriology 

E.  Duclaux,  Traite  de  Microbiologie,  1898-1901. 

F.  Lafar,  Handbuch  der  Technischen  Mykologie,  1903-1915. 

F.  Lohnis,  Handbuch  der  Landwirtschaftlichen  Bakteriologie,  1910. 

Gino  de  Rossi,  Microbiologia  Agraria  e Tecnica,  1921-1922. 

D.  Books  on  Bacteriological  Technique  and  Diagnostics. 

F.  D.  Chester,  Manual  of  Determinative  Bacteriology,  1901. 

E.  B.  Fred,  Laboratory  Manual  of  Soil  Bacteriology,  1916. 

C.  Gunther,  Einfuhrung  in  das  Studium  der  Bakteriologie,  1906. 


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P.  G.  Heinemann,  Laboratory  Guide  in  Bacteriology,  1911. 

K.  B.  Lehmann  und  R.  0.  Neumann,  Bakteriologische  Diagnostik,  1920. 
F.  Lohnis,  Laboratory  Methods  in  Agricultural  Bacteriology,  1913. 

E.  Periodicals 

Abstracts  of  Bacteriology . 

Centralblatt  fur  Bakteriologie,  I.  und  II.  Abteilung. 

Journal  of  Bacteriology . 

Journal  of  Dairy  Science. 

Soil  Science. 


Part  I 

GENERAL  MORPHOLOGY  AND  PHYSIOLOGY  OF 
BACTERIA  AND  RELATED  MICROORGANISMS 


CHAPTER  I 


MORPHOLOGY  OF  BACTERIA  AND  RELATED 
MICROORGANISMS 

Morphological  and  physiological  characters  of  cultivated  plants  and 
domesticated  animals  determine  the  degree  of  usefulness  of  these 
organisms.  Therefore  such  knowledge  is  of  fundamental  importance 
to  the  agriculturist.  The  same  holds  true  with  regard  to  bacteria 
and  other  microorganisms  useful  or  harmful  to  agriculture.  Because 
most  of  these  organisms  can  be  seen  clearly  only  with  a very  powerful 
microscope,  a discussion  of  their  morphological  features  will  help  in 
gaining  an  accurate  understanding  of  their  peculiar  nature,  which  is  at 
the  root  of  their  surprisingly  great  activity. 

Form  and  Size  of  Cells. — While  all  higher  plants  and  animals  repre- 
sent very  complicated  and  finely  adjusted  structures  of  cells  and  cell 
compounds,  it  is  the  single  cell  that  acts  as  the  living  unit  as  far  as  bac- 
teria, yeasts,  molds,  and  protozoa  are  concerned.  When  these  single  cells 
grow  and  multiply,  it  often  happens,  of  course,  that  temporarily  a num- 
ber of  cells  will  be  more  or  less  closely  connected.  Especially  the  lower 
fungi  (molds)  frequently  form  threads  or  chains  of  cells,  which  some- 
times may  be  seen  with  the  naked  eye.  But  here  again  the  single  cell 
remains  the  living  unit ; long  threads  break  up  into  short  joints,  so-called 
oidia,  which  process  can  be  clearly  observed  with  the  common  white  mold 
( Oidium  lactis)  frequently  visible  as  a white  fur-like  cover  on  sour  cream. 

The  most  characteristic  forms  of  bacteria,  lower  fungi,  and  protozoa 
are  pictured  on  Plate  I.  The  shape  of  the  single  cell  is  fundamentally 
the  same  as  in  higher  organisms : globular,  oval,  cylindrical,  or  spiral. 
There  are  smaller  or  larger  differences  and  variations  in  every  case ; and 
intermediate  shapes  between  ovals  and  short  rod-forms,  as  well  as  between 
cylindrical  and  spiral  cells,  so-called  comma-shaped  organisms,  are  not 
infrequent.  Generally  the  cells  of  yeasts,  molds,  and  protozoa  are  con- 
siderably larger  than  the  bacteria,  but  also  this  rule  has  its  exceptions. 
Azotobacter,  shown  in  Fig.  5,  Plate  I,  one  of  the  most  important  soil 
organisms,  reaches,  for  instance,  a rather  conspicuous  size,  while  some- 
times yeasts  and  protozoa  may  remain  much  smaller.  Usually  special 
treatments — staining  with  aniline  dyes,  or  mixing  the  unstained  cells 

15 


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with  India  ink — are  applied  to  get  a clearer  picture  than  is  obtainable 
with  the  living  cells  suspended  in  water. 

How  incredibly  small  bacteria  really  are,  will  become  clear  from  the 
following  consideration.  At  1000-fold  magnification  many  of  the  rod- 
shaped bacteria  measure  about  0.5  by  1.75  mm.  A man  magnified  on  the 
same  scale  would  appear  as  a giant  1700  meters  tall  and  500  meters 
broad.  Between  such  an  immense  being  and  men  of  normal  size 
exactly  the  same  relation  in  size  would  exist  as  between  men  and  the 
bacteria  1000-fold  magnified.  Therefore,  to  reach  an  accurate  concep- 
tion of  the  real  size  of  the  bacteria,  another  step  of  the  same  relation 
would  have  to  be  made,  but  this  is  almost  beyond  imagination. 

Measuring-  Bacteria. — On  account  of  their  minute  size  bacteria  and 
other  microorganisms  are  measured  by  “micro-millimeters.  ” One  micro- 
millimeter,  usually  abbreviated  “micron”  (plur.  micra)  and  written 
1/x,  is  equivalent  to  1/1000  mm.  Most  of  the  globular  bacteria,  usually 
called  cocci,1  have  a diameter  of  about  1/4.  The  short  rod  forms,  as  they 
are  found,  for  instance,  in  the  root  nodules  of  leguminous  plants,  measure 
usually  1/2-3AyO--^1/2lx,  while  the  long  rods  reach  4 to  6/4  or  more  in 
length.  Among  all  the  bacteria  thriving  in  milk,  butter,  cheese,  manure, 
and  soil,  only  a few  will  be  found  smaller  than  1 4/4  or  larger  than  10/4. 
Instances  of  exceptionally  large  bacteria  are  found  in  the  case  cf  organ- 
isms connected  with  the  transformation  of  sulfur  compounds  (Chapter 
VII,  5)  ; their  length  may  reach  40  to  60/x  or  more.  The  cells  of  the 
causative  agent  of  pleuropneumonia  of  cattle,  on  the  other  hand,  measure 
only  0.1  to  0.2/4.  After  the  ultra-microscope  wTas  discovered,  some  authors 
were  of  the  opinion  that  they  had  found  a special  group  of  “ultra- 
microorganisms,” much  smaller  than  the  smallest  bacteria  known.  Un- 
doubtedly  such  very  minute  forms  exist,  but  it  seems  as  if  they  are 
merely  peculiar  growth  types  of  larger  bacteria  or  of  protozoa. 

Size  and  Efficiency. — Single  cells  of  yeasts,  molds,  and  protozoa 
are  usually  5-  to  10-  to  20-fold  larger  than  bacteria.  For  two  reasons 
these  differences  are  of  great  physiological  importance.  First,  the  smaller 
a body,  the  greater  the  area  of  its  surface  in  relation  to  its  volume.  Sec- 
ond, as  the  exchange  of  substances  in  most  of  the  metabolic  processes, 
caused  by  bacteria  or  fungi,  takes  place  through  the  cell  wall,  the  relative 
size  of  its  surface  naturally  determines  to  a large  extent  the  efficiency 
of  the  active  cell.  Figure  5 shows  why  the  smaller  size  of  the  bacteria 

i Derived  from  6 k6kkos  (kokkos)  = fruit  kernel.  Strictly  taken,  the  term  globular 
bacteria  should  not  be  used,  because  the  name  bacterium  comes  from  fSc Urpov  (baktron) 
or  paKTrjpia  (bakteria),  Greek  words  for  rod.  But  at  present  the  term  bacteria  is  so 
generally  used  for  all  of  these  organisms,  quite  irrespective  of  their  shape,  that  it  has 
practically  lost  its  original  meaning. 


Lohnis-Fred,  Text  book 


Plate  I 


1-4.  Globular  bacteria,  stained  with  methylene  blue,  1000 
1.  Micrococci  2. =3.  Streptococci  from  milk  4.  Sarcina 


5-8.  Rod-shaped  bacteria,  stained  with  fuchsin,  X 1000 
5.  Azotobacter  6.  Nodule  bacteria  7.  Bact.  casei  8.  Hay  bacillus 


9-12.  Curved  and  spiral  bacteria,  India  ink  preparations,  X 1000 
9.  Proteus  10.  Vibrio  sp.  11.  Spirillum  sp.  12.  Spirochaeta  sp. 


13-16.  Yeasts,  molds,  and  protozoa,  living,  X 1000 
13-14.  yeasts  of  different  shape  15.  Oidium  lactis  16.  Protozoa 


MORPHOLOGY  OF  BACTERIA  AND  RELATED  MICROORGANISMS  17 

renders  them  more  efficient  than  are  the  larger  fungus  cells.  Rectangle 
A represents  the  surface  of  a cube  whose  length  of  edge  is  1 mm.  If  the 
calculation  is  simplified  by  ascribing  to  bacteria  and  fungi  cubical  shape 
and  an  average  size  of  1/a3  and  of  10/a3  respectively,  it  follows  that  the 
1 mm.  cube  will  be  filled  by  1 million  fungus  cells  or  by  1000  million 
bacteria  cells.  The  total  surface  of  1 million  10/a  cubes  is  600  mm.2  (rec- 
tangle B),  that  of  1000  million  1/a  cubes,  however,  is  6000  mm.2  (rec- 
tangle C) . A 100-  or  1000-fold  reduction  in  size  results  in  a 100-  or  1000- 
fold  enlargement  of  the  active  surface  and  also — at  least  to  a certain 
extent — of  the  efficiency  of  these  organisms. 


Fig.  5. — Rectangle  A:  Surface  of  a cube  whose  length  of  edge  is  1 mm.  B:  Total 
surface  of  1 million  cubes  whose  length  of  edge  is  10/a.  C : Total  surface  of  1000 
million  cubes  whose  length  of  edge  is  l/i. 

One  thousand  million  cells  within  1 cubic  millimeter,  about  the  size 
ox  a pin  head,  is  again  something  well  beyond  imagination.  If  five  men 
would  each  count  two  cells  per  second  throughout  every  one  of  300  days 
in  a year,  they  would  finish  within  this  period  not  more  than  100  millions, 
which  is  only  one-tenth  of  the  total  sum. 

Size  and  Number. — Considering  the  minute  size  of  the  bacteria  it  is 
easily  understood  why  such  large  numbers  of  them  may  be  present  in 
soil,  water,  air,  food,  etc.  Figure  6 shows  three  glass  containers  (1/10 
original  size)  which  were  filled  with  20  kg.  milk,  butter,  and  Swiss  cheese, 
respectively.  In  the  centers  of  the  front  panes  small  cubes  of  black 
glass  were  fastened,  indicating  how  much  space  would  be  filled  by  the 
bacteria  present  in  that  amount  of  milk,  butter,  or  cheese,  if  they  all 
could  be  collected  in  these  places. 


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Wherever  bacteria  display  great  activity,  rod-shaped  cells  are  most 
prevalent.  This  again  is  easily  understood,  when  it  is  taken  into  account 
that  the  active  surface  is  comparatively  much  larger  with  a rod  than  with 
a globule.  But  globular  cells  are  most  frequent  among  the  bacteria  in  the 
air ; there  is  little  chemical  activity,  and  small  globules  are  naturally  bet- 
ter able  than  rods  to  float  in  the  air  for  a long  time. 

Variability  of  the  Cell  Form. — It  is  a well  known  fact  that  the  form 
of  higher  organisms  always  varies  to  some  extent,  as  is  especially  notice- 
able with  cultivated  plants  and  domesticated  animals.  Therefore,  it  is 


Fig.  6— Glass  containers  with  milk,  butter,  and  cheese  (xV  orig.  size).  Germ  content 
in  milk  2^  millions  per  c.c.;  in  butter  20  millions  per  g.;  in  cheese  500  millions 

per  g. 

not  surprising  that  the  small  single  cells  of  bacteria  ana  related  organ- 
isms exhibit  similar  tendencies.  And  if  one  considers  how  much  more 
they  are  exposed  to  the  modifying  influences  of  their  environment,  it  will 
at  once  become  obvious  that  even  much  greater  variations  of  the  cell 
forms  are  to  be  expected  in  these  cases. 

Figure  9 on  Plate  I presents  a bacterium,  especially  inclined  to  assume 
various  shapes,  which  on  account  of  this  behavior  has  been  named 
Proteus,  in  memory  of  the  old  Homeric  sea-god,  of  whom  it  was  told  that 
he  could  change  into  every  form  imaginable.  But  on  closer  examination 
the  nodule  bacteria  (Fig.  6,  Plate  I),  as  well  as  the  streptococci 1 taken 
1 A chain  of  beads,  used  as  necklace,  was  called  by  the  Greeks  ffrperrbs  (streptos). 


MORPHOLOGY  OF  BACTERIA  AND  RELATED  MICROORGANISMS  19 


from  milk  (Figs.  2 and  3,  Plate  I),  also  display  certain  variations  in  their 
cell  forms  and  the  cocci  some  deviation  from  the  typically  globular  shape. 

But  the  alterations  of  bacterial  cell  forms  are  not  restricted  to  such 
comparatively  small  variations.  Figure  1 on  Plate  II  indicates  what 
changes  may  occur  with  the  slender  straight  rods  of  Bacterium  casei, 
shown  in  Fig.  7 on  Plate  I.  Figure  2 on  Plate  II  should  be  compared 
with  Fig.  6 on  Plate  1,  and  Figs.  3 and  4 on  Plate  II  with  Fig.  8 on 
Plate  I (the  normal  form  of  Bacillus  Malabarensis  is  very  similar  to  that 
of  the  hay  bacillus). 

Involution  Forms. — Cells  of  atypical  shape  are  often  termed 
“involution  forms,”  and  this  name  is  indeed  quite  appropriate  as  far  as 
such  changes  are  really  due  to  cell  degeneration.  Involution  is  contrary 
to  evolution,  and  synonymous  with  degeneration,  that  is  retrograde  devel- 
opment leading  to  death.  Unfortunately,  it  has  become  a very  wide- 
spread habit  to  speak  of  involution  forms  wherever  a type  of  growth  be- 
comes visible  which  the  observer  considers  to  be  atypical.  The  opinion 
that  bacteria  must  be  always  globules,  rods,  or  spirals,  and  that  only 
these  are  typical  or  “legitimate”  forms,  has  so  firmly  taken  hold  of  so 
many  bacteriologists  that  it  is  usually  considered  entirely  superfluous  to 
make  a thorough  investigation  of  the  viability  and  the  further  behavior 
of  such  assumed  involution  forms.  But  whenever  such  investigations 
were  made,  it  was  frequently  discovered  that  these  changed  cells  were  by 
no  means  pathological  or  in  course  of  degeneration. 

The  irregular  cell  forms  of  the  nodule  bacteria  appear,  for  instance, 
when  development  is  at  its  height,  and  they  are  very  active  in  fixing 
nitrogen  from  the  air.  Other  bacteria,  participating  in  the  process  of 
nitrogen  fixation,  display  similar  changes.  And  increased  knowledge 
has  shown  that  pathogenic  organisms,  too,  may  appear,  while  fully  active, 
in  shapes  widely  differing  from  those  often  called  typical. 

Monomorphism  and  Pleomorphism. — After  Robert  Koch  had  de- 
veloped his  methods  of  isolating  the  bacteria  and  of  growing  them  in  pure 
cultures,  it  was  soon  discovered  that  under  constant  conditions  a con- 
spicuous uniformity  in  growth  was  to  be  observed.  This  fact  was  con- 
trary to  earlier  opinion.  It  had  been  thought  before  that  bacteria,  like 
fungi  and  protozoa,  were  able  to  assume  many  different  forms;  they  all 
were  considered  to  be  polymorphous  or  pleomorphous.1  But  as  the  bac- 
teria thus  far  studied  were  practically  all  grown  in  mixed  cultures,  and 
the  new  results,  recorded  by  R.  Koch  and  his  pupils,  apparently  proved 
without  exception  that  pure  cultures  of  bacteria  did  not  display  such 

1 Derived  from  iro\6s  (polys)  = many,  tt\Icov  (pleon)  = more,  and  txopfp-q  (morphe)  = 
form. 


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pleomorphism,  the  opposite  point  of  view  gained  great  strength  among 
bacteriologists.  The  bacteria  were  now  declared  to  be  strictly  mono- 
morphous, and  the  theory  of  monomorphism  was  taught  nearly  every- 
where ; only  comparatively  few  bacteriologists  did  not  accept  it. 

However,  as  more  and  more  data  accumulated,  it  became  increasingly 
difficult  to  reconcile  the  facts  observed  with  this  theory.  "When  the  ex- 
periments were  conducted  under  strictly  uniform  and  constant  conditions, 
as  a rule,  uniform  and  constant  results  were  secured.  But  exceptions  were 
not  entirely  absent,  and  changes  in  the  environmental  conditions  led  to 
still  greater  deviations.  By  declaring  one  type  of  growth  in  each  case  to 
be  typical,  and  by  discarding  other  forms  as  ‘ ‘ atypical  ’ ’ or  as  signs  of  ‘ ‘ in- 
volution, ” the  monomorphistic  dogma  could  and  can  be  preserved  for  a 
while.  But  if  the  facts  are  weighed  impartially,  no  doubt  remains  that 
like  lower  fungi,  algae,  and  protozoa,  which  have  long  been  known  to  be 


pleomorphous,  the  bacteria  too  are  able  to  assume  different  cell  forms  in 
the  course  of  their  full  development,  although  under  constant  conditions 
uniformity  and  constancy  are  frequently  observed. 

Cell  Compounds ; Branched  Growth. — With  all  lower  organisms  the 
single  cell  is  the  living  unit,  but  cell  compounds  may  be  temporarily 
formed  by  bacteria,  and  with  the  fungi  this  type  of  growth  is  more  fre- 
quent and  more  permanent.  The  globular  cells  of  the  cocci  may  occur  in 
tetrads  or  in  irregular  clusters  (Fig.  1,  Plate  I),  in  which  case  they  are 
sometimes  called  staphylococci,1  or  in  short  or  long  chains  as  streptococci 
(Figs.  2 and  3,  Plate  I),  or  in  regular  cubical  bundles,  made  up  of  8,  16, 
32,  or  more  cells  (Fig.  4,  Plate  I),  to  which  the  name  Sarcina  is  usually 
applied.2  Compounds  of  rod-shaped  cells  are  either  chains  or  threads ; in 
the  chain  the  single  units  are  still  easily  discernible,  while  in  the  threads 
the  dividing  cell  walls  are  less  clearly  visible  or  have  entirely  vanished. 


Fig.  7. — Chain  of  budding 
yeast  cells  (X500). 


Fig.  8. — Branching  thread 
of  a mold  ( X500). 


1 Derived  from  <rTa<pv\i?i  (staphyle)  = bunch  of  grapes. 

2 Crosswise  tied  baggage  was  called  sarcina  by  the  Romans. 


Lohnis -Fred,  Text  book 


Plate  II 


1-4.  Branched  growth  and  gonidangia,  stained  with  fudisin,  X 1000 
1.  Bact.  casei  2.  Nodule  bacteria  3-4.  Bacillus  Malabarensis 


5-8.  Bacteria  with  flagella,  stained  after  Ermengem,  X 1000 
5.  Nitrosomonas  6.  B.  fluorescens  7.  B.  radicicola  8.  Proteus 


9-10.  Bacleria  with  slime  11-12.  Bacteria  with  spores 

stained,  X 1000  stained,  X 1000 

9.  Nodule  bacteria  10.  Leuconostoc  11.  B.  amylobacter  12.  B.  putrificus 


13-16.  Yeasts  and  molds  with  spores,  living 
13.  Sporulating  yeast  14.  Mucor  15.  Aspergillus  16.  Penicillium 

X 600  X 300  X 300  X 300 


MORPHOLOGY  OF  BACTERIA  AND  RELATED  MICROORGANISMS  21 


Figures  7 and  8 illustrate  these  differences,  and  they  show  at  the  same 
time  how  lateral  outgrowth  may  lead  to  branched  forms. 

Figures  1 and  2 on  Plate  II  picture  branched  bacteria,  which  according 
to  the  monomorphistic  doctrine  were  often  classed  as  involution  forms. 
But  sufficient  evidence  is  available  at  the  present  time  that  branching 
occurs  in  many  bacteria,  and  very  probably  in  all  of  them.  Even 
species  which  usually  grow  in  globular  shape  may  change  to  irregular 
branching  growth ; and  it  is  especially  noticeable  that  branched  forms 
are  found  more  frequently  in  young  than  in  old  cultures,  quite  contrary 
to  what  is  to  be  expected  from  true  involution  forms.1 

Structure  of  Cells. — The  same  relation  which  exists  with  regard  to 
cell  morphology  between  bacteria,  lower  fungi,  and  protozoa  on  the  one 
side,  and  the  cells  of  higher  organisms  on  the  other,  becomes  apparent 
when  the  inner  structure  of  those  cells  is  examined.  Again  there  is 
analogy  in  principle,  but  greater  simplicity  in  the  minute  bacterial 
cells.  Their  very  small  size  makes  such  cytological  studies,  of  course,  ex- 
ceptionally difficult,  and  it  is  not  surprising  that  the  results  secured  by 
different  investigators  are  not  always  in  good  agreement.2  The  methods 
used  in  such  studies,  especially  the  various  modes  of  fixing  and  staining 
the  cells,  cause  frequently  more  or  less  profound  alterations  of  the  deli- 
cate structures,  and  vexatious  artefacts  are  often  formed.  In  most  cases 
a highly  complicated  protoplasm  represents,  in  size  as  in  importance,  the 
major  part  of  these  minute  cells,  and  this  is  usually  surrounded  by  a 
more  or  less  solid  membrane  ; only  some  of  the  protozoa  are  without  mem- 
brane, and  therefore  characterized  by  their  very  changeable  forms. 

Protoplasm. — Rarely  does  the  protoplasm  present  a homogeneous 
appearance ; more  frequently  it  is  distinctly  granular,  as  shown  in  Figs. 
13  to  16  on  Plate  I,  and  in  Fig.  7 in  the  text.  Part  of  these  granules  are  so- 
called  cell  inclusions,  representing  either  reserve  material  (fat,  glycogen, 
volutin,  etc.)  or  foreign  bodies  (for  instance  bacteria,  taken  up  as  food 
by  the  protozoa),  or  they  are  growing  reproductive  organs,  which  are  lib- 
erated when  fully  developed.  Whether  or  not  cell  nuclei  are  present 
among  these  cell  granules,  has  been  a long  disputed  question,  and  some 
authors  are  still  firmly  convinced  that  the  bacteria  at  least  have  no 
nuclei.  It  is  beyond  dispute  that  in  all  microorganisms  no  such  highly 
organized  nuclei  are  to  be  found  as  in  the  cells  of  higher  plants  and  ani- 
mals. But  nuclear  substances  are  undoubtedly  present,  either  finely 

1 Detailed  references  concerning  the  pleomorphism  of  the  bacteria  (also  on  their 
branched  growth)  may  be  found  in  F.  Lohnis’  “Studies  upon  the  Life  Cycles  of  the 
Bacteria,”  Memoirs  Nat.  Acad.,  vol.  XVI,  No.  2,  1921. 

2 These  cytological  problems  are  thoroughly  discussed  in  A.  Meyer,  “ Die  Zelle  der 
Bakterien,”  and  in  Ch.  Marshall’s  “Microbiology.” 


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«. 


distributed  as  so-called  chromidia,  or  concentrated  into  one  or  more 
globular  or  rod-shaped  bodies.  In  protozoa  and  in  lower  fungi,  vacuoles 
are  often  visible  within  the  protoplasm,  while  bacterial  cells  are  nearly 
always  completely  filled  with  active  protoplasm,  which  fact  furnishes 
another  reason  for  their  comparatively  very  high  efficiency. 

Cell  Wall. — The  membrane  surrounding  the  protoplasm  is  always 
more  or  less  slimy,  and  agglomerates  of  bacteria  are  therefore,  as  a rule, 
soft,  viscous,  and  sometimes  very  sticky.  Such  slimy  bacteria  prove 
occasionally  troublesome  in  milk,  bread,  and  in  sugar  solutions.  When 
examined  under  the  microscope  in  living  condition  such  cells  are 
surrounded  by  a bright  halo  of  varying  thickness  (Figs.  13  to  16,  Plate 
I),  while  drying  and  staining  usually  furnishes  pictures  like  Figs.  3 and  9 
on  Plate  II.  The  slime  has  shrunk  in  these  cases,  leaving  an  empty 
space  around  the  stained  cells ; sometimes  the  cells  themselves  are  found 
lying  outside  these  empty  spaces.  If  the  slime  is  more  solid  it  will  be 


Fig.  9. — Anthrax  bacilli  with  capsules  (X1000). 

stained,  too ; Fig.  10  on  Plate  II  illustrates  such  a case,  representing  the 
so-called  Leuconostoc,  a slime  producing  streptococcus,  sometimes  found 
in  sugar  factories.  A term  frequently  used  for  slimy  agglomerates  of 
this  kind  is  “ zoogloea” ; it  means  really  nothing  else  than  “animal 
slime,  ’ ’ and  should  be  avoided. 

Sometimes  the  slimy  layer  around  the  bacterial  cell  becomes  very 
solid  and  forms  a so-called  capsule,  which  is  accessible  only  to  special 
staining  methods.  The  presence  of  such  capsules  is  very  characteristic 
for  certain  species ; it  is  used  for  instance  in  diagnosing  anthrax  bacilli 
in  the  blood  of  diseased  animals  (Fig.  9).  If  bacteria  growing  in  long 
threads  surround  themselves  with  such  a solid  cover,  the  term  sheath  is 
used  instead  of  capsule. 

Plasmolysis  and  Plasmoptysis. — Under  normal  conditions  the  proto- 
plasm is  firmly  pressing  against  the  cell  wall,  but  sudden  changes  in  the 
concentration  of  the  solution  surrounding  the  cell  may  cause  conspicuous 
alterations.  If  there  is  an  increase  in  osmotic  pressure  at  the  outside  of 
the  cell,  water  escapes  and  the  protoplasm  shrinks  accordingly,  loosening 


MORPHOLOGY  OF  BACTERIA  AND  RELATED  MICROORGANISMS  23 


itself  from  the  cell  wall.  In  the  opposite  ease  too  much  water  may  enter 
the  cell,  so  that  the  wall  breaks  under  the  strain,  and  the  protoplasm 
is  thrown  out  as  a drop  with  more  or  less  force.  The  first  process  is 
called  plasmolysis,  the  second  one  plasmoptysis,1  and  it  was  mentioned  in 
the  historical  review  that  such  enforced  alterations  have  been  observed 
very  early  by  Edm.  King.  More  recently  both  terms  have  been  some- 
times used  rather  incorrectly  for  explaining  various  occurrences,  which 
in  fact  have  nothing  to  do  with  such  purely  physical  effects. 

Motility. — If  living  bacteria  are  examined  under  the  microscope, 
suspended  in  a drop  of  water  or  nutrient  solution,  some  of  them  show 
active  motility,  while  others  do  not.  Yeasts  and  molds  are  all  immotile, 
protozoa  on  the  other  hand  nearly  always  motile,  except  their  resting 
forms.  But  it  is  not  always  easy  to  decide  accurately  whether  certain 
bacteria  are  actively  motile  or  not.  Very  small  corpuscles  of  the  size  of 
bacteria,  as  for  instance  the  black  particles  of  India  ink  suspended  in 
water,  exhibit  also  some  locomotion,  which  however  is  entirely  passive, 
of  course.  It  is  the  so-called  Brownian  movement  which  keeps  the  mole- 
cules of  liquids  permanently  in  a state  of  unrest  and  also  causes  a con- 
tinuous swinging  movement  of  suspended  minute  bodies.  If  bacteria  of 
great  motility  are  tested,  no  error  can  be  made ; but  with  slow  moving 
forms  much  care  is  needed  to  reach  a correct  conclusion.  It  is  due  to 
this  fact  that  the  literature  contains  many  uncertain  or  incorrect  state- 
ments with  regard  to  the  motility  of  bacteria.  Young  cultures  naturally 
are  best  suited  for  such  tests. 

The  speed  of  locomotion  varies  greatly.  Some  bacteria  travel,  as  men 
do,  1 to  li/2  their  own  length  per  second,  others  are  5 to  10  times  faster ; 
but  fast  flying  birds  reach  the  50-fold  of  their  own  length  in  the  same 
time.  The  type  of  bacterial  movement  also  shows  much  variation.  It 
may  be  straight,  or  wiggling,  or  gyrating.  Short  rods  often  tumble 
about,  appearing  temporarily,  when  in  an  upright  position,  like  small 
cocci.  Spiral  forms  are  whirling  like  ships’  propellers.  The  aspect  be- 
comes especially  lively,  when  in  a drop  of  putrid  liquid  (old  liquid 
manure,  for  instance)  the  motile  bacteria  are  chased  around  by  bacteria- 
hunting  protozoa. 

Flagellation. — The  organs  of  locomotion  of  the  motile  bacteria  are 
in  nearly  all  cases  very  thin,  whip-like  protrusions  of  the  cell  wall,  so- 
called  cilia  or  flagella.  Only  some  spiral  forms  (classed  as  spirochaetes) 
have  instead  of  flagella  an  undulating  membrane,  which  serves  the  same 
purpose.  Because  of  their  extreme  thinness  bacterial  cilia  can  be  clearly 
seen  only  after  having  been  treated  in  some  special  manner.  Generally 


1 Derived  from  XiW  (lyein)  = loosen,  and  from  irrieiv  (ptyein)  = spit. 


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the  staining  of  bacterial  flagella  needs  much  patience  and  care,  and  many 
special  methods  have  been  recommended  for  this  purpose.  Well  made 
preparations  show  that  the  cilia  are  to  be  found  either  all  around  the 
cell,  or  only  at  the  ends  singly  or  in  tufts  (Figs.  5 to  8,  Plate  II).  Terms 
frequently  used  for  these  different  types  of  flagellation  are  peritrichous, 
polar  or  cephalotrichous,  monotrichous  and  lophotrichous.1  Cilia  are 
easily  thrown  off,  they  may  then  agglomerate  to  wavy  braids,  which  are 
sometimes  mistaken  for  spirilla  or  spirochaetes. 

Active  motility  increases,  of  course,  the  efficiency  of  the  bacteria. 
They  can  quickly  travel  to  places  where  organic  substances  await  de- 
struction, and  even  fairly  dry  soil  contains  enough  water  to  allow  free 
passage  to  these  minute  organisms. 

1 Derived  from  irepl  (peri)  = around,  rpl\^  (triches)  = hairs,  K'tpaXr)  (kephale)  =head. 
and  \6<t>os  (lophos)  = tuft. 


CHAPTER  II 


DEVELOPMENT  OF  BACTERIA  AND  RELATED  MICRO- 
ORGANISMS 


For  propagating  higher  plants,  vegetative  parts  of  the  mother  plant 
(cuttings,  tubers)  may  be  used,  or  new  growth  is  secured  from  seeds,  that 
is  from  special  reproductive  organs  of  sexual  origin.  The  same  two  ways 
are  open  for  the  lower  organisms,  although  the  purely  vegetative  multi- 
plication of  cells  is  much  more  frequent.  The  simple,  but  efficient  struc- 
ture of  the  vegetative  cells  of  the  bacteria  favors  this  kind  of  develop- 
ment. 

Multiplication  of  Vegetative  Cells. — After  a cell  has  reached  its  full 
size,  it  divides  into  two  cells,  these  when  grown  sufficiently,  separate  into 
four,  four  into  eight,  and  so  on.  Because  of  this  simple  fission  the  bac- 
teria have  been  called  schizomycetes,  which  means  fission  fungi,1  although 
fundamentally  the  same  mode  of  cell  multiplication  takes  place  in  all 
lower  as  well  as  in  the  higher  organisms.  Quite  unique,  however,  is  the 
rapidity  of  multiplication  of  bacterial  cells.  Under  suitable  conditions  a 
new  fission,  or  a doubling  of  the  cells  takes  place  after  20  to  30  minutes, 
and  if  this  would  go  on  in  the  same  manner  for  a day  or  two,  the  follow- 
ing stupendous  multiplication  would  result : 


One  bacterium  would  produce 

after  1 hour  4 bacteria 

after  2 hours  16  bacteria 

after  3 hours  64  bacteria 

after  8 hours  65,536  bacteria  (in  round  figures  60,000) 

after  15  hours  1000  million  = approximately  1 mm.3 

after  23  hours  65,000  mm.3  = 65  cm.3 

after  35  hours  1000  million  cm.3  = 1000  m.3 


Therefore,  the  possibility  exists  that  the  progeny  of  one  single  bac- 
terial cell  represents  after  iy2  days  of  steady  multiplication  a bacterial 
mass  that  would  fill  200  trucks  of  5 tons  capacity  each.  It  is  self-evident 
that  natural  conditions  will  never  allow  such  excessive  multiplication. 
Lack  of  food,  the  detrimental  effects  of  metabolic  products,  the  antagonis- 
tic action  of  other  organisms,  and  various  other  influences  will  always 


1 Derived  from  <rx^eiv  (schizein)  = split,  and  fuffojs  (mykes)  = fungus. 

25 


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cheek  this  rapid  development  after  a comparatively  short  time.  For  a 
while,  however,  those  theoretical  possibilities  may  indeed  become  realities, 
and  this  is  the  reason  why  occasionally  bacterial  growth  will  appear  and 
spread  with  an  almost  miraculous  speed.  Exact  determinations  have 
shown,  for  instance,  that  a small  number  of  bacteria  planted  in  milk, 
actually  increased  according  to  the  following  scale : 1 

In  2 3 4 5 6 hours 

At  12.5°  C.  4-  6-  8-  26-  435-fold 

36.0°  C.  23-  60-  245-  1830-  3800-fold 

With  one  fission  every  30  minutes  the  multiplication  would  have 
been : 16-,  64-,  256-,  1024-  and  4096-fold.  This  shows  that  milk,  kept  at 
high  temperature,  permits  indeed  an  extremely  rapid  bacterial  growth. 

Sooner  or  later  this  rapid  increase  is  always  followed  by  an  equally 
rapid  decrease.  The  following  bacterial  counts  of  a sample  of  milk  may 
serve  as  an  illustration : 2 

At  the  beginning  After  3 6 12  18  24  hours 

Bacteria  in  millions  per  cc. . . 0.37  12.75  226  8070  32,243  2286 

In  the  case  of  f ungi  and  protozoa  cell  multiplication  is,  as  a rule,  gen- 
erally slower,  but  it  may  continue  for  a much  longer  time.  Nevertheless, 
it  is  a well-known  fact  that  for  instance  food  of  slightly  acid  reaction 
(sour  cream,  cottage  cheese,  fruit  jam,  etc.)  may  be  overrun  by  molds, 
especially  in  warm,  moist  weather,  in  a comparatively  very  short  time. 
A few  invisible  cells  grow  up  to  a mass  clearly  visible  to  the  naked  eye. 

Formation  of  Colonies. — On  solid  or  semi-solid  substrates  (cheese, 
sour  cream,  etc.)  bacterial  and  fungous  growths  at  first  appear  in  the 
shape  of  more  or  less  regular  circular  discs,  which  are  called  colonies. 
Colonies  of  molds  are  made  up  of  a radiate  network  of  threads,  the  so- 
called  mycelium,3  which  can  be  distinguished  even  by  the  naked  eye  from 
the  smooth,  paste-like  colonies  of  yeasts  and  bacteria.  In  spoiled  jellies 
especially,  the  latter  are  sometimes  clearly  visible  as  small,  whitish  or 
yellowish,  globular  or  lens-shaped  bodies,  approximately  of  the  size 
of  millet  kernels.  Because  in  such  substrates  the  bacteria  are  unable  to 
make  use  of  their  motility,  and  accordingly  the  progeny  of  one  cell  will 
develop  to  a colony  at  the  place  where  the  original  germ  was  located, 
semi-solid  transparent  jellies  have  become  a very  helpful  means  for  cul- 
tivating and  studying  bacteria. 

In  order  to  obtain  an  accurate  knowledge  of  the  nature  and  activity  of 

1 Cnopf,  Centralbl.  f.  Bakt.,  vol.  6,  1889,  p.  553. 

2 Budinoff,  Centralbl.  f.  Bakt.,  II  Abt.,  vol.  34,  1912,  p.  177. 

3 Derived  from  jut ikt/s  (mykes)  =fungus,  and  rpvos  (helios)  =sun. 


DEVELOPMENT  OF  BACTERIA  AND  RELATED  MICROORGANISMS  27 

the  different  bacteria,  it  is  absolutely  necessary,  of  course,  to  isolate  them 
and  to  investigate  each  kind  separately.  It  is  possible,  but  very  difficult, 
to  pick  out  single  bacterial  cells  under  the  microscope.  More  commonly 
bacteriologists  make  use  of  transparent  gelatinous  media  of  such  composi- 
tion that  these  are  solid  at  low,  but  liquid  at  a higher  temperature,  which, 
however,  is  not  so  high  as  to  kill  the  bacteria.  If  these  are  then  evenly 
distributed  in  the  liquefied  material,  and  this  is  spread  out  and  solidified 
in  a flat,  covered  glass  dish,  usually  called  Petri  dish,  the  ensuing  growth 
presents  itself  in  a manner  more  or  less  similar  to  that  pictured  in  Fig.  1 
on  Plate  III. 

The  filamentous  colonies  of  molds  can  easily  be  distinguished  from 
the  more  compact,  whitish,  gray,  or  yellow  colonies  of  bacteria,  some  of 
which  have  caused  a greenish  or  brown  discoloration  of  the  nutrient  gela- 
tine. A few  colonies  have  liquefied  the  substrate  around  them  and  are 
slowly  dispersing  in  the  liquid ; others  are  completely  imbedded,  and 
appear  as  those  small,  whitish,  grain-like  colonies  mentioned  before.  Col- 
onies of  yeasts  cannot  be  differentiated  by  the  naked  eye  from  those  of 
bacteria.  The  small  pinkish  colony  in  the  foreground  to  the  right  was, 
for  instance,  made  up  of  yeast  cells. 

Microscopic  Appearance  of  Colonies. — At  a comparatively  low  mag- 
nification, the  differences  among  the  various  colonies  become  much  more 
prominent,  as  may  be  seen  from  Figs.  2 to  5 on  Plate  III.  The  coarse 
granulation  of  the  yeast  colony  (Fig.  4)  is  due,  of  course,  to  the  relatively 
large  size  of  its  cells,  and  the  fine  rhizoid  threads  of  the  mold  (Fig.  5) 
are  equally  conspicuous. 

If  a thin  cover  glass  is  slightly  pressed  for  a moment  against  a bac- 
terial colony,  then  carefully  lifted,  and  stained  according  to  one  of  the 
methods  commonly  used  in  the  bacteriological  laboratory,  very  instructive 
contad-preparates  are  obtained,  which  furnish  an  accurate  picture  of  the 
bacteria  as  they  are  situated  in  the  colony,  because  practically  all  cells 
from  the  surface  of  the  colony  have  stuck  to  the  glass,  if  there  was 
enough,  but  not  too  much  pressure.1 

Variation  in  Colony  Formation. — Giant  colonies  of  bacteria  and 
fungi  may  be  secured  if  care  is  taken  that  each  colony  is  surrounded  by 
a sufficiently  large  area  of  nutrient  substrate  whicli  is  kept  free  from  all 
other  growth,  as  is  shown  in  the  small  glass  containers  (so-called  Soyka- 
flasks)  pictured  in  Fig.  6 on  Plate  III.  The  concentric  growth  always 

1 Good  photographs  of  contact  preparates  of  bacterial  colonies  may  be  found  in  a 
paper  by  C.  Axelrad,  Zeitschr.  f.  Hyg.,  vol.  44,  1903,  p.  477.  Detailed  discussions  of 
colony  formation  were  published  by  H.  B.  Hutchinson,  Centralbl.  f.  Bakt.  II.  Abt., 
vol.  17,  1906-07,  p.  65,  and  by  Fr.  Ors6s,  Centralbl.  f.  Bakt.  I.  Abt.  Orig.,  vol.  54, 
1910,  p.  289. 


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noticeable  with  such  colonies,  is  clearly  represented  by  the  zones  visible 
in  the  giant  colony  of  Penicillium.  Similar  zones  may  also  be  seen  with 
bacterial  colonies;  changing  environmental  influences  (temperature, 
light,  etc.)  are  responsible  for  them.  In  nature  the  concentric  growth 
of  immense  fungous  colonies  finds  its  expression  in  the  so-called  fairy 
rings ; the  uneven  growth  of  the  grass  indicates  at  such  places  the  pro- 
gressive development  and  the  following  decay  of  the  mycelium  in  the 
soil,  which  then  exerts  a fertilizing  influence. 

The  appearance  of  bacterial  and  fungous  colonies  varies,  of  course, 
according  to  the  conditions  under  which  they  develop.  However,  under 
practically  uniform  conditions  the  pictures  presented  by  growing  colonies 
are  remarkably  constant,  characteristic,  and  of  considerable  diagnostic 
value.  This  regularity  and  persistence  is  especially  surprising  if  we 
keep  in  mind  that  with  these  simple  microorganisms  the  single  cell  repre- 
sents an  independent  living  unit.  But  the  same  tendency  of  association 
which  induces  bees,  ants,  and  other  higher  organisms  to  unite  and  to 
enter  into  complicated,  well-characterized  organizations,  which  survive 
the  single  organisms,  seems  to  be  active  even  in  these  very  first  steps  of 
organic  development ; the  protozoa  alone  are  an  exception  to  this  rule. 

If  the  microorganisms  are  growing  in  liquids  the  colony  formation  is 
less  conspicuous,  especially  with  motile  bacteria.  Nevertheless,  also  in 
such  cases  characteristic  agglomerations  may  become  visible,  provided 
that  the  liquid  substrate  remains  undisturbed.  Of  special  interest  are 
the  so-called  bacterial  “niveaux”  or  “plates,”  which  may  develop  in 
putrid  liquids.1  In  the  glass  cylinder  shown  at  the  left  side  in  Plate  IX 
such  a “plate”  of  sulfur  bacteria  is  seen  floating  in  the  middle  of  the 
cylinder ; curious  appendices  are  hanging  dowm  into  the  lower  part  of 
the  solution,  whose  nature  will  be  discussed  in  Chapter  VII,  5. 

Conjunction,  Conjugation,  and  Copulation. — If  well  made  contact 
preparates  from  bacterial  colonies  are  carefully  inspected,  or  if  young 
living  bacterial  cells  are  closely  examined  under  the  microscope,  many 
cells  may  be  seen  which  are  connected  with  each  other  by  thin  lateral 
bridges  (Fig.  10),  or  by  beak-like  protrusions  very  similar  to  those  occur- 
ring with  lower  fungi  and  algae.  In  the  latter  case  it  is  beyond  doubt 
that  a primitive  sexual  process  is  taking  place  between  the  united  cells, 
which  is  usually  termed  conjugation.  With  the  minute  bacteria  the 
uniting  bridges  are,  of  course,  much  less  conspicuous.  For  a long  time 
very  little  attention  was  paid  to  these  facts,  but  enough  observations  are 
available  at  present  to  indicate  that  this  conjunction  of  bacterial  cells  is 

1 Bei.ierinck,  Centralbl.  f.  Bakt.,  vol.  14,  1893,  p.  827;  Jegunow,  Centralbl.  f.  Bakt. 
II.  Abt.,  vol.  2,  1896,  p.  13;  Lehmann  und  Curchod,  1.  c.  vol.  14,  1905,  p.  449. 


Lohnis-Fred,  Text  book 


Plate  III 


1.  Colonies  of  bacteria  and  fungi  in  Petri  dish 

2/3  nat.  size 


2-5.  Microscopic  appearance  of  colonies,  X 50 

2.  Bact.  coli  3.  B.  fluorescens  4.  Pink  yeast  5.  Peniciilium 


6.  Giant  colonies  of  bacteria  and  fungi  in  Soyka  flasks 

2/3  nat.  size 


DEVELOPMENT  OF  BACTERIA  AND  RELATED  MICROORGANISMS  29 


of  similar  physiological  importance  as  the  conjugation  or  the  copulation 
of  other  microorganisms.1  The  last  named  term  is  usually  reserved  for 
those  cases  where  a complete  fusion  of  two  cells  takes  place,  as  is  com- 
mon especially  with  protozoa. 

Formation  of  Reproductive  Organs. — In  the  case  of  higher  organ- 
isms as  a rule  sexual  processes  precede  the  production  of  reproductive 
organs.  In  principle  the  same  holds  true  with  regard  to  the  lower  or- 
ganisms, but  sexual  differentiation  as  well  as  sexual  intercourse  is 
much  less  conspicuous  in  this  case.  Frequently  reproductive  organs  are 
produced  by  bacteria,  fungi,  and  by  protozoa  undoubtedly,  or  very  prob- 
ably, in  an  asexual  way.  Up  to  the  present,  many  investigators  have 
thought  that  the  reproductive  organs  of  the  bacteria  were  always  of 
asexual  origin.  The  very  inconspicuous  mode  of  conjunction  was  usually 


Fig.  10. — Large  sulfur  bacteria  (Chroma-  Fig.  11. — Sporulating  threads  of  the  hay 

tium)  in  conjunction  (X750).  bacillus  unstained  (living)  X1000. 

overlooked.  Careful  observation  reveals,  however,  that  at  first  (usually 
during  the  first  four  days)  many  cells  are  to  be  found  in  the  conjunct 
stage,  and  that  the  formation  of  reproductive  organs  becomes  prominent 
only  after  this  period  has  passed.  Nevertheless,  it  is  not  to  be  doubted 
that  many  reproductive  organs  are  produced,  indeed,  asexually. 

Bacterial  Endospores. — Best  known,  most  characteristic,  and  most 
important  are  the  so-called  endospores  of  the  bacteria.  They  are  pre- 
eminently resting  forms,  well  foi’tified  against  unfavorable  influences, 
and  destined  to  preserve  bacterial  life  over  periods  of  drought,  etc., 
when  vegetative  life  becomes  impossible.  Old  hay,  for  instance,  always 
contains  numerous  spores  of  the  so-called  hay  bacillus  ( B . subtilis). 
If  water  is  added  to  the  hay  the  spores  germinate  to  new  rods  and 
threads;  later  new  spores  are  formed  therein  (Fig.  11),  which  again 
survive  the  dying  cells.  Except  in  a few,  rather  rare  cases,  only  one 

1 Lohnis,  “Studies  upon  the  Life  Cycles,  etc.,”  Mem.  Nat.  Acad.,  XVI,  No.  2, 
Chap.  IV. 


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spore  is  formed  in  each  cell;  therefore  bacterial  endospores  do  not  par- 
ticipate in  the  multiplication  of  cells,  although  such  a statement  has 
been  made  repeatedly.  Not  all  bacteria  are  able  to  produce  endospores; 
only  a few  fairly  well  characterized  groups  have  this  ability,  which  how- 
ever may  be  lost  temporarily  or  permanently. 

When  the  spore  is  being  formed  a contraction  and  concentration  of 
nuclear  and  cytoplastic  material  takes  place,  the  newly  formed  globular 
or  ovoid  body  surrounds  itself  with  a rather  solid  membrane,  and  the 
rest  of  the  cell  gradually  fades  away.  In  some  cases  the  diameter  of 
the  growing  spore  is  distinctly  larger  than  the  width  of  the  mother  cell ; 
accordingly,  the  rod-form  of  the  latter  changes  to  a club-like  or  drum- 
stick-like appearance  (Figs.  11  and  12,  Plate  II).  Cells  of  such  forms 
are  often  called  Clostridium  and  plectridium,  respectively.1  Due  to  their 
low  water  content,  ripe  spores  are  not  as  easily  stained  as  vegetative  cells 


Fig.  12. — Germination  of  bacterial  spores  (above)  and  of  mold  spores  (below). 

(Fig.  11,  Plate  II)  ; many  special  staining  methods  have  been  devised 
which  permit  a differential  and  very  characteristic  staining  of  cells  and 
of  spores  (Fig.  12,  Plate  II). 

The  germination  of  these  spores  usually  begins  with  a more  or  less 
pronounced  swelling,  then  the  new  germ  breaks  forth  either  in  polar, 
equatorial,  or  oblique  position  (Fig.  12),  and  the  membrane  of  the 
spore  is  either  left  behind,  or  it  is  again  (though  less  frequently)  used  in 
making  the  new  cell  wall.  Some  authors  believed  that  the  different  modes 
of  germination  (polar,  equatorial,  or  oblique)  could  serve  for  diagnostic 
purposes.  However,  they  are  not  sufficiently  constant  to  be  accepted 
as  safe  marks  of  distinction. 

Other  Reproductive  Organs  of  the  Bacteria. — Besides  endospores 
four  other  kinds  of  reproductive  organs  are  produced  by  bacteria  : Micro- 
cysts,  arthrospores,  gonidia,  and  regenerative  bodies.2 

1 Derived  from  k\w<ttt]p  (kloster)  = spindle,  and  tt\9)ktpov  (plektron)  = Greek  instru- 
ment for  striking  the  lyre. 

2 Lohnis,  “Memoir,”  Chap.  II. 


DEVELOPMENT  OF  BACTERIA  AND  RELATED  MICROORGANISMS  31 


Microcysts  1 are  resting  forms,  frequently  produced  by  bacteria  of 
globular  or  oval  shape,  simply  by  a thickening  and  hardening  of  the  cell 
wall.  The  Azotobacter  cells  shown  in  Fig.  5 on  Plate  I illustrate  this 
occurrence. 

Arthrospores 2 are  to  be  found  especially  in  certain  rod-shaped 
bacteria.  These  rods  divide  into  several  short  roundish  joints,  each  of 
which  surrounds  itself  with  a fairly  resistant  cell  wall,  and  assumes  the 
character  of  a resting  cell. 

Gonidia  3 are  small  round  bodies,  relatively  conspicuous  in  some  large 
thread-like  forms  (iron  bacteria),  but  also  produced  by  all  other  bacteria. 
In  the  latter  case  usually  1 to  4 of  them  are  to  be  found  in  each  cell; 
their  minute  size  is  responsible  for  their  being  very  little  known.  They 
are  not  resting  forms,  but  are  mostly  motile,  and  may  multiply  as  such 
before  growing  up  to  new  regular  cells.  Therefore,  they  are  able  to  par- 
ticipate considerably  in  the  multiplication  of  the  bacteria.  Frequently 
they  develop  while  still  inclosed  in  the  mother  cell,  becoming  buds  and 
branches  in  this  case.  Occasionally  they  are  produced  in  greater  num- 
bers than  four,  in  which  case  the  mother  cell  undergoes  an  inflation  and 
develops  to  a gonidangmm,  that  is  a giant  cell  of  globular,  club,  pear, 
or  spindle-shaped  appearance,  which  was  often  seen  but  usually  dis- 
carded as  an  involution  form  (Figs.  3 to  4,  Plate  II). 

Regenerative  bodies  are  also  globular,  but  larger  than  the  gonidia, 
and  have  firmer  cell  walls.  When  free,  they  look  like  micrococci 
and  are  able,  like  these  or  the  gonidia,  to  multiply  as  such  before  repro- 
ducing normal  cells.  They  are,  in  fact,  an  intermediate  step  between 
gonidia  and  normal  cells ; sometimes  they  are  also  motile.  Occasionally 
they  appear  exactly  like  the  zygospores  of  fungi  and  algae  at  the  point 
where  two  conjunct  cells  have  united  (Fig.  1 on  Plate  II). 

Reproductive  Organs  of  Fungi  and  of  Protozoa. — Among  the  vari- 
ous reproductive  organs  of  the  lower  fungi  only  the  following  ones  may 
be  mentioned  here: 

Spores,  produced  within  sporangia,  as  shown  in  Figs.  13  to  14  on 
Plate  II  for  a yeast  and  for  a very  common  mold,  named  Mucor,  serving 
as  resting  cells  as  well  as  for  multiplication. 

Conidia ,4  produced  in  great  numbers  at  the  end  of  conidiophores  of 
various  shapes,  playing  an  important  role  in  the  almost  ubiquitous  dis- 
tribution of  such  molds  as  PeniciTlium  and  Aspergillus  (Figs.  15  to  16, 
Plate  II).  They  cover  the  mycelia  more  or  less  completely  (Fig.  6,  Plate 

1 Derived  from  /oVtis  (kystis)  =bag. 

2 Derived  from  &p8pov  (arthron)  = joint. 

3 Derived  from  y6ros  (gonos)  = offspring. 

4 Derived  from  Kovla  (konia)  = dust. 


32 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


III),  and  at  the  slightest  disturbance  they  rise  like  a cloud  of  dust  into 
the  air,  where  they  remain  floating  for  a long  time.  Germination  of  conidia 
and  mold  spores  starts  as  with  bacterial  spores  (Fig.  12),  but  not  in- 
frequently a multiple  germination  takes  place. 

Chlamydospores  and  gemmae,1  formed  in  somewhat  analogous  man- 
ner to  bacterial  arthrospores,  and  like  these  representing  resting  forms. 
When  germinating,  the  chlamydospores  produce  fertile  branches,  the 
gemmae  vegetative  growth. 

The  protozoa,  too,  may  either  encapsulate,  forming  a cyst,  or  by 
segmentation  may  produce  several  spores  or  sporozoites.  The  former  are 
typical  resting  forms,  the  latter  means  of  multiplication  and  reproduc- 
tion. 

Autolysis  and  Symplastic  Stage. — Sooner  or  later,  especially  when 
the  food  supply  becomes  insufficient,  many  cells  of  bacteria,  fungi,  and 


Fig.  13.  Fig.  14.  Fig.  15. 

Fig.  13. — Dissolution  of  normal  Azotobacter  cells  (X1000). 

Fig.  14. — Symplasm  (X1000). 

Fig.  15. — Regenerative  bodies  growing  from  the  symplasm  (X1000). 

protozoa  may  dissolve,  or — according  to  a frequently  used  expression— 
autolysis  may  take  place.  This  may  mean  death  to  the  organisms,  but 
by  no  means  always.  If  the  observations  are  continued,  new  development 
may  become  visible  in  these  amorphous  residues,  and  new  cells  may  be 
evolved  similar  to  or  different  from  those  of  the  preceding  generation. 
Figures  13  to  15  show  these  steps  in  the  development  of  Azotobacter. 

If  the  amorphous  product  of  autolysis  is  still  alive,  usually  vigorous 
inner  movements  are  noticeable,  and  sometimes  also  a slow,  amoeboid, 
creeping  locomotion.  Sooner  or  later  minute  globoid  bodies,  so-called 
regenerative  units,  become  distinguishable  which  either  by  direct  up- 
growth or  by  fusion  of  several  units  reproduce  new  cells.  At  first  re- 
generative bodies  often  appear  (Fig.  15)  like  those  produced  by  vegeta- 

1 Derived  from  xXajuh  (chlamys)  = cloak;  gemma  = bud. 


DEVELOPMENT  OF  BACTERIA  AND  RELATED  MICROORGANISMS  33 

tive  cells ; or  besides  the  normal  cells  others  of  various  irregular  shape 
may  appear,  formerly  as  a rule  incorrectly  classed  as  involution  forms. 

This  melting  together  of  the  contents  of  numerous  cells,  the  thorough 
mixing  of  the  amorphous  material,  and  the  re-arrangement  of  it  into 
new  cells  are  undoubtedly  of  great  importance  for  the  continuity  of 
microbial  life  in  unfavorable  environment  and  for  its  adaptation  to  new 
activity  under  changed  conditions.  This  phase  in  the  development  of 
bacteria  and  related  microorganisms  has  been  called  the  symplastic  stage, 
and  the  product  of  the  fusion  of  the  dissolved  cells  was  named  symplasm.1 
Occasionally  the  latter  assumes  globular  shape,  surrounds  itself  with  a 
fairly  solid  membrane,  and  becomes  a macrocyst.  Such  macrocysts  have 
been  found  with  nitrifying  bacteria,  sulfur  bacteria,  and  others. 

1 Derived  from  <rvv  (syn-,  before  p:  sym-)  = together,  and  irX&atreiv  (plassein)  = build- 


CHAPTER  III 


CLASSIFICATION  OF  BACTERIA,  FUNGI,  AND  PROTOZOA 

At  present  bacteria,  lower  fungi,  and  protozoa  are  only  partly  known. 
New  kinds  are  discovered  nearly  every  day,  but  the  descriptions  given 
are  often  very  incomplete.  Since  the  middle  of  the  nineteenth  century 
it  has  been  generally  acknowledged  that  the  full  development,  the  com- 
plete “life  cycle,”  of  a fungus  or  of  a protozoon  must  be  thoroughly 
investigated  before  such  an  organism  can  be  properly  named  and  cor- 
rectly classified.  Before  that  time  many  so-called  species  were  proposed 
which  later  proved  to  be  merely  stages  in  the  life  cycles  of  other  organ- 
isms. Accordingly  numerous  lower  fungi  have  received  several  names 
and  have  been  changed  in  their  systematic  positions  repeatedly.  Due  to 
the  monomorphistic  dogma,  which  predominated  in  bacteriology  for  sev- 
eral decades,  the  life  cycles  of  these  microorganisms  are  practically  un- 
known at  present,  and  their  classification  is,  therefore,  now  in  the  same 
position  as  was  that  of  the  lower  fungi  about  fifty  years  ago. 

Artificial  and  Natural  Classification. — When  Linnaeus  began  to  clas- 
sify plants  and  animals  upon  a scientific  basis,  he  had  to  rely  mostly  on 
more  or  less  complete  descriptions  of  their  morphology.  In  this  way  a 
so-called  artificial  classification  was  secured,  which  was  later  replaced 
by  more  natural  classifications  founded  on  a more  adequate  knowledge  of 
morphology  as  well  as  of  physiology  and  of  the  natural  relationship  of 
these  organisms.  With  fungi  and  protozoa  the  same  change  in  classify- 
ing has  taken  place  more  recently,  and  is  still  going  on.  With  the  bac- 
teria, however,  only  an  artificial  classification  is  possible  at  present  be- 
cause of  lack  of  knowledge  concerning  their  complete  life  histories.  It  is 
true  that  besides  morphological  characters,  physiological  data  are  also 
frequently  used  in  classifying  bacteria.  Occasionally  arrangements  ob- 
tained in  this  manner  are  termed  “natural”  classifications,  but  they  too 
are  purely  artificial. 

For  agricultural  purposes  a more  practical  grouping  is  usually  quite 
sufficient,  viz.,  to  classify  bacteria  and  related  microorganisms  according 
to  their  activity,  for  instance  as  lactic  acid,  or  butyric  acid  producing, 
nitrifying,  nitrogen  fixing  organisms,  etc.  Undoubtedly  this  point  of  view 

34 


CLASSIFICATION  OF  BACTERIA,  FUNGI , AND  PROTOZOA 


35 


is  of  greatest  importance  to  agriculturists,  while  the  other  question  con- 
cerning artificial  or  natural  classification  is  among  the  tasks  to  be  solved 
by  the  bacteriologists. 

Scientific  and  Common  Nomenclature. — According  to  the  rules  of 
scientific  nomenclature  first  promulgated  by  Linnaeus  and  now  generally 
accepted,  each  group  of  organisms  of  practically  uniform  character  and 
clearly  distinct  from  others,  should  receive  a double  name  in  Latin,  the 
first  word  indicating  the  genus,  the  second  one  the  species.  Triticum 
sativum  and  Solarium  tuberosum  are  examples  of  scientifically  correct 
species  denomination. 

With  respect  to  microorganisms  the  situation  is  much  less  satisfactory, 
due  (1)  to  the  incomplete  knowledge  of  their  characters,  (2)  to  the  in- 
clination of  some  authors  to  rearrange  and  rename  all  that  has  been 
classified  and  named  before,  (3)  to  the  tendency  of  many  authors  to 
bestow  names  that  are  not  in  accordance  with  those  accepted  rules. 
Sometimes  whole  descriptions  are  given  instead  of  binomial  names,  for 
instance  Streptococcus  acidi  paralactici  non  liquefaciens  Halensis,  Hashi- 
moto;  or  Granulobacillus  saccharobutyricus  immobilis  liquefaciens, 
Grassberger  et  Schattenfroh.  Several  authors  have  renamed  bacteria 
and  other  microorganisms  merely  because  the  new  names  seemed  to 
them  more  appropriate.  Furthermore,  many  so-called  species  have 
been  introduced  into  the  literature  despite  the  absence  of  adequate  de- 
scriptions, and  the  same  species  name  has  been  repeatedly  given  to  quite 
different  organisms.  To-day  considerable  experience  is  necessary  in 
order  to  get  a clear  view  of  the  whole  situation,  and  much  remains  to  be 
done  before  the  scientific  nomenclature  and  classification  of  the  bacteria 
will  be  in  a fairly  satisfactory  condition. 

Besides  scientific  determinations  many  common  names  are  in  use  with 
lower  as  well  as  with  higher  organisms.  The  agriculturist  speaks  of  wheat 
and  potatoes,  instead  of  Triticum  sativum  and  Solarium  tuberosum,  and  in 
the  same  manner  such  common  names  as  lactic  acid  bacteria,  nitrifying 
bacteria,  hay  bacillus,  tubercle  bacillus,  etc.,  are  frequently  used  and 
quite  sufficient  in  many  cases. 

Classification  of  Bacteria. — The  following  simple  arrangement  of  a 
comparatively  small  number  of  genera  has  been  used  by  European  bac- 
teriologists during  the  last  forty  or  fifty  years : 

f 1.  Singly,  in  pairs,  tetrads, 

or  clumps,  not  in  chains . Micrococcus 

I.  Cells  mostly  globular,  rarely  rod-like  { 2.  In  pairs  or  in  chains Streptococcus 

I 3.  In  regular  bundles  of  8,  16, 

(,  or  more  cells Sarcina 


36 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


II.  Cells  mostly  rod-like,  rarely  globular 
or  curved 


1.  Without  endospores Bacterium 

2.  With  endospores Bacillus 


III.  Cells  mostly  curved  or  spiral,  rarely 
globular  or  rod-like 


1.  Of  comma-shape Vibrio 

2.  Of  rigid  spiral  shape Spirillum 

3.  Of  flexible  spiral  shape ....  Spirochaeta 


This  classification  rests  entirely  on  a morphological  basis,  and  on 
account  of  the  small  number  of  genera  many  species  had  to  be  in- 
corporated into  each  genus.  Nevertheless  the  system  was  widely  adopted 
and  has  proved  very  helpful.  Most  thorough  work  on  it  was  done  by  K. 
B.  Lehmann,  professor  of  hygiene  at  the  University  of  Wurzburg,  Ger- 
many, who  published  excellent  descriptions  of  numerous  species  all  based 
on  his  own  careful  experimental  studies.1  Morphological  as  well  as 
physiological  features  are  both  fully  considered,  and  on  this  basis  a clear 
arrangement  into  well  defined  groups  was  made. 

In  America  not  this,  but  another  system  of  German  origin  was 
adopted,  proposed  about  25  years  ago  by  IT.  Migula,2  but  almost  unani- 
mously rejected  by  the  European  bacteriologists.  Relatively  unimpor- 
tant and  highly  variable  features,  such  as  flagellation,  pigmentation,  type 
of  germination  of  spores,  etc.,  were  used  for  defining  genera  and  species. 
Furthermore,  the  whole  arrangement  was  based  mostly  on  very  incom- 
plete descriptions  made  by  other  authors,  and  only  to  a very  limited  ex- 
tent upon  original  experimental  work. 

More  recently  S.  Orla-J ensen,  professor  at  Copenhagen,  Denmark, 
proposed  another  system,  which  is  almost  exclusively  based  on  more  or 
less  uncertain  biochemical  facts  and  hypotheses.3  Despite  the  author’s 
claim,  it  represents  by  no  means  a “natural”  system.  Nevertheless,  since 
the  serious  defects  of  Migula ’s  arrangement  have  led  to  its  abandon- 
ment also  in  America,  the  proposition  made  by  Orla-Jensen  is  now 
looked  upon  with  much  favor  in  this  country. 

A Committee  of  the  Society  of  American  Bacteriologists  adopted 
several  parts  of  this  latest  system,  combined  them  with  others  taken  from 
Migula ’s  and  other  authors’  classifications,  and  recommended  the  result- 
ing compilation  for  general  use.4  The  number  of  genera  was  consider- 
ably increased,  but  at  least  most  of  the  families  into  which  these  genera 
were  united  agree  well  with  the  older  and  better  arrangement  mentioned 


1 Lehmann  und  Neumann,  “Atlas  und  Grundriss  der  Bakteriologie,”  6th  ed.,  1920. 

2 Migula,  “ System  der  Bacterien,”  1897-1900. 

3 Orla-Jensen,  “ Das  natiirliche  Bakterien-System,”  Centralbl.  f.  Bakt.,  II.  Abt., 
vol.  22,  1909,  pp.  305-346. 

4 C.-E.  A.  Winslow,  J.  Broadhurst,  R.  E.  Buchanan,  Ch.  Krumwlede,  Jr., 
L.  A.  Rogers  and  G.  H.  Smith,  “The  families  and  genera  of  the  bacteria,”  Jour,  of 
Bad.,  vol.  V,  No.  3,  1920,  pp.  191-229. 


CLASSIFICATION  OF  BACTERIA,  FUNGI,  AND  PROTOZOA 


37 


above.  These  are  I.  Coeeaceae,  II.  Bacteriaceae,  III.  Baeillaeeae,  and  IV. 
Spirillaceae.  In  addition  to  these,  two  other  families  are  proposed  by  the 
Committee:  Pseudomonadaceae  (with  one  genus  Pseudomonas  Mig.)  and 
Nitrobacteriaceae  (with  several  genera,  based  on  biochemical  behavior). 

The  genus  name  Pseudomonas,  originally  introduced  by  Migula  and 
still  frequently  used  by  American  bacteriologists,  was  intended  to  desig- 
nate cells  with  polar  flagella.  It  is  beyond  dispute  that  the  same  organ- 
ism, for  instance  Azotobacter,  may  have  peritrichous  or  polar  flagella, 
and  that  closely  related  species,  for  instance  among  the  nodule  bacteria, 
may  show  these  two  types  of  flagellation.  It  was  pointed  out  that  this 
differentiation  is  especially  unsuitable  for  scientific  classification.  But  as 
the  term  Pseudomonas  is  again  revived  in  the  classification  proposed  by 
the  Committee  of  the  Society  of  American  Bacteriologists,  its  meaning 
should  be  known  at  least.  It  remains  to  be  added  that  the  terms 
Bacterium  and  Bacillus  are  also  used  in  quite  a different  manner  by  the 
followers  of  Migula : Bacterium  for  immotile  rods,  Bacillus  for  those 
with  peritrichous  flagellation.  Occasionally  the  same  generic  names  find 
still  other  application ; but  this  is  of  little  importance  in  all  those  cases 
where  the  species  name  is  quite  distinct,  as  it  always  should  be.  The  ab- 
breviation B.,  as  in  B.  radiciola,  B.  eoli,  B.  fluoreseens,  etc.,  may  then 
mean  Bacterium  or  Bacillus;  the  species  name  clearly  indicates  what 
organism  is  meant. 

Classification  of  Lower  Fungi. — Only  comparatively  few  groups  of 
lower  fungi  take  part  in  the  processes  to  be  discussed  in  agricultural  bac- 
teriology. They  are  known  as  yeasts  and  as  molds. 

The  yeasts  (Figs.  13  and  14,  Plate  I,  text  Fig.  7)  are  usually  classed 
as  Saccharomyces  if  they  produce  endospores  (Fig.  13,  Plate  II),  as 
Torula  if  they  are  non-sporulating,  and  as  Mycoderma  if  they  are  grow- 
ing as  tough  membranes  on  the  surface  of  nutrient  solutions. 

Among  the  so-called  molds  the  following  five  genera  are  of  interest : 

Oidium  or  Oospora,  characterized  by  its  transparent  fragile 
mycelium,  composed  of  short  oidia  or  conidia  (Fig.  15,  Plate 
I)  ; Oidium  lactis  is  a most  common  species,  regularly  present 
on  sour  cream,  etc. 

Dematium,  of  similar  appearance  as  Oidium,  but  with  dark 
colored  mycelium. 

Mucor,  with  non-septate,  richly  branched  mycelium,  large 
sporangia  (Fig.  14,  Plate  II),  and  occasional  formation  of 
zygospores. 

Aspergillus,  with  septate  mycelium  and  clubbed  conidiophores 
(Fig.  15,  plate  II). 


38 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


Pencillium,  with  septate  mycelium  and  brush-shaped  conidio- 
phores  (Fig.  16,  Plate  II).  A rare  type  of  fructification 
sometimes  found  with  this  and  the  preceding  genus  is  the 
so-called  perithecium  (a  type  of  sporangium). 

Classification  of  Protozoa. — Three  groups  of  protozoa  are  fairly 
constant  inhabitants  of  water  and  soil,  and  occasionally  very  active  as 
destroyers  of  bacterial  life.  They  are  classed  as : 

Rhizopoda  or  Sarcodina,1  motile  by  pseudopodia.  Amoebae  and 
lleliozoa  are  wide-spread  representatives  of  this  group. 

Mastigophora,2  with  long  whip-like  flagella,  of  which  the  Flagel- 
lates are  most  common. 

Ciliates  or  Infusoria,  covered  by  a fur  of  short  fine  cilia  used  for 
locomotion  as  well  as  for  securing  food. 

Relation  of  Bacteria  to  Fungi  and  to  Protozoa. — Early  investigators 
classed  the  bacteria  because  of  their  motility  among  the  animals  (as 
“animaleula”  or  “infusoria”).  At  present  they  are  mostly  considered 
to  be  plants.  In  fact,  however,  neither  of  the  popular  terms  plant  and 
animal  is  well  applicable  to  these  lowest  organisms.  They  are  better  left 
in  a class  by  themselves,  or  they  may  be  united  with  all  other  unicellular 
organisms  under  the  term  Protista,  as  recommended  by  the  well-known 
zoologist  E.  Haeckel. 

Motility  and  cell  structure  of  the  bacteria  show  many  features  com- 
mon with  protozoa  as  well  as  with  lower  algae.  Other  characters  re- 
semble those  of  lower  fungi,  and  since  it  was  discovered  that  all  bacteria 
are  able  to  grow  in  branched  forms,  several  authors  thought  that  they 
should  be  classed  as  fungi.3  There  is  especially  one  group,  the  so-called 
Mycobacteriaceae,  showing  at  least  temporarily  distinctly  fungoid 
growth,  and  another  one,  the  Actinomycetes,  standing  exactly  on  the 
border-line  between  bacteria  and  fungi.  Many  scientific  and  practical 
reasons,  however,  are  clearly  against  such  a merging  of  bacteria  and 
fungi.  It  is  undoubtedly  best  to  retain  the  bacteria  as  a separate  group 
of  organisms,  showing  certain  relations  to  lower  fungi,  protozoa,  and 
lower  algae,  but  being  different  from  all  of  them  in  other  respects. 

1 aapKd iSijs  (sarkodes)  means  fleshy,  referring  to  their  particular  appearance.  The 
first  name  means  root-footed  (plt;a  [rhiza]  =root,  and  ttovs  [pous]  =foot). 

2 Derived  from  /xdo-rd  (mastix)  = whip,  flagellum,  and  <plpeiv  (pherein)  = carry,  bear. 

3E.  Bergstrand,  “On  the  Nature  of  Bacteria,”  Jour.  Infect.  Diseases,  vol.  27 

No.  1,  1920,  pp.  1-22. 


CHAPTER  IV 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT 

It  was  pointed  out  before  that  the  variability  in  form,  in  motility, 
and  in  other  characters,  so  widespread  among  the  bacteria,  is  largely  de- 
pendent on  the  conditions  under  which  they  are  living.  But  in  nature 
the  environmental  conditions  are  not  constant,  nor  do  the  organisms  al- 
ways react  in  the  same  manner.  A more  or  less  speedy  adaptation  to  dif- 
ferent environmental  conditions  is  frequently  noticeable,  and  in  this 
respect  much  more  profound  alterations  are  possible  among  the  micro- 
organisms, than  are  known  among  higher  plants  and  animals.  The  fun- 
damental reason  for  this  is  that  within  a few  days  several  hundred  gener- 
ations of  bacteria  may  follow  each  other.  Accordingly,  changes  in  the 
living  conditions  may  lead  to  conspicuous  alterations  of  the  bacterial 
characters  within  a few  weeks  or  months,  while  decades,  centuries,  or 
still  longer  periods  would  pass,  before  by  similar  progressive  adaptations 
of  the  succeeding  generations  analogous  transformations  in  the  appear- 
ance and  behavior  of  higher  organisms  could  be  realized.  Furthermore, 
it  is  easily  understood  that  single  cells  of  relatively  simple  structure  are 
much  more  responsive  to  changed  environmental  conditions  than  are 
complicated  organisms,  built  up  of  myriads  of  highly  specialized  cells. 


1.  BACTERIAL  NUTRITION 

Bacteria  and  other  microorganisms  obtain  their  food  directly  or  in- 
directly by  leading  either  a saprophytic 1 or  a parasitic  life.  But  these 
differences  are  by  no  means  constant.  So-called  parasitic  bacteria  are 
cultivated  in  the  laboratory  on  artificial  substrates;  and  also  under 
natural  conditions,  they  may  grow  as  saprophytes  especially  in  milk  and 
in  stable  manure.  On  the  other  hand,  if  circumstances  are  suitable, 
originally  saprophytic  organisms  may  invade  living  plants  and  animals 
and  so  become  parasites. 

Some  of  the  bacteria  can  live,  like  green  plants,  on  purely  inorganic 
substances,  while  most  of  them  have  to  depend  on  organic  nutrients  like 
animals.  In  the  literature  the  former  are  not  infrequently  mentioned  as 

1 Derived  from  <rairp6s  (sapros)  = putrid,  and  <pvrbv  (phyton)  = plant. 

39 


40 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


autotrophic  or  prototrophic  organisms,  the  latter  as  metatrophic  or  hetero- 
trophic.  But  as  most,  if  not  all,  of  the  so-called  autotrophic  bacteria 
are  also  able  to  live  in  heterotrophic  style,  it  is  preferable  to  avoid  these 
terms  as  well  as  those  first  mentioned. 

Chemical  Composition  of  Cells. — The  composition  of  higher  organ- 
isms varies  according  to  their  nutrition.  But  these  variations  are  again 
much  greater  with  bacteria  than  with  cells  of  higher  plants  and  animals. 

Vegetative  cells  of  bacteria  and  fungi  contain  large  amounts  of  water 
(75  to  98  per  cent)  like  young  growing  plants.  Spores  have  much  less 
(about  40  per  cent),  because  they  consist  mostly  of  concentrated  plas- 
matic substances.* 1  Accordingly,  the  specific  weight  of  bacterial  cells  is 
usually  close  to  and  a little  above  1.0,  while  that  of  spores  is  distinctly 
higher,  approximately  1.3-1.4.2 

High  or  low  percentages  of  mineral  substances  are  found  (2  to  30  per 
cent  in  the  dried  cells)  according  to  the  kind  of  nutrition.  What  varia- 
tions are  possible  in  this  respect,  even  with  the  same  species,  may  be  seen 
from  the  following  data.  Cholera  bacteria  wrere  found  to  contain  in  their 
dry  substance  :3 


Per  Cent 


Grown  in  Grown  in 

Beef  Broth  Mineral  Solution 


Organic  substances 
Mineral  substances 


f nitrogenous. . 
I nitrogen-free 


68 

6 

26 


36 

50 

14 


The  nitrogen  content  of  bacteria  and  fungus  cells  is  usually  high 
(about  10  per  cent  of  the  dry  substance),  but  sometimes  it  is  found  to  be 
as  low  as  1 per  cent.  Again  the  same  species  may  show  wide  variations; 
for  instance,  in  one  case  1.33  per  cent,  in  another  12.8  per  cent  N were 
found  in  Azotobacter  cells.4  The  inclination  of  this  species  to  produce 
sometimes  large  quantities  of  slimy  substances,  which  are  free  of  nitrogen, 
was  probably  the  foremost  reason  for  the  differences  observed.  Besides 
the  protoplasm  of  the  cells,  the  cell  walls  of  bacteria  and  fungi  may  also 
contain  nitrogenous  substances,  among  which  chitin  is  best  known. 
“Chromatin”  and  “volutin”  are  designations  for  two  groups  of  nitro- 

1 Kruse,  ‘ Allgemeine  Mikrobiologie,”  1910,  p.  53. 

2 Lohnis,  “Handbuch  der  landwirtschaftlichen  Bakteriologie,”  1910,  p.  261. 

3 E.  Cramer,  Archiv.f.  Hyg.,  vol.  16,  1893,  p.  151;  vol.  22.  1895,  p.  167. 

4 Gerlach  und  Vogel,  Centralbl.  f.  Bakt.,  II.  Abt.  Bd.  9,  1902,  p.  884;  C.  Hoffmax 
and  B.  W.  Hammer,  Centralbl.  f.  Bakt.,  II.  Abt.  Bd.  28,  1910,  p.  137. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  41 


genous  cell  inclusions,  whose  occurrence  is  frequently  studied  in  micro- 
chemical tests. 

Carbohydrates  are  much  less  common  in  the  cells  of  microorganisms 
than  in  those  of  higher  plants.  Cellulose  is  mostly  absent,  and  sugar  as 
well  as  starch  is  usually  replaced  by  glycogen  and  granulose.  Fatty  and 
waxy  substances  may  be  present  in  considerable  quantities,  especially  in 
the  spores  of  bacteria  and  fungi,  but  also  in  the  vegetative  cells  of  cer- 
tain species.  In  the  dried  cells  of  the  tubercle  bacillus,  for  instance,  40 
per  cent  fat  has  been  found.1  The  presence  of  fat  or  wax  in  the  cell 
walls  makes  such  cells  hard  to  stain.  The  generally  used  aqueous  stain- 
ing solutions  remain  without  effect  in  such  cases ; this  was  the  reason  why 
the  discovery  of  the  tubercle  bacilli  was  not  made  at  an  earlier  time. 

Nitrogen  Requirement. — Practically  all  nitrogenous  substances  can 
serve  as  food  for  one  or  another  group  of  bacteria  and  fungi,  which  be- 
cause of  this  fact  play  such  an  important  role  in  the  transformation  of 
nitrogen  in  nature. 

Generally,  proteins  represent  the  best  sources  of  nitrogen.  Meat,  for 
instance,  is  very  liable  to  be  attacked  by  myriads  of  putrefying  bacteria ; 
in  the  laboratory,  milk  and  peptone  solutions  are  used  for  cultivating 
numerous  species. 

Amides  and  amino  acids  are,  as  a whole,  less  suitable,  although  some 
of  them,  especially  asparagin  and  aspartic  acid,  can  also  be  used  in  many 
cases.  Others,  like  urea,  uric  and  hippuric  acids,  are  utilized  only  by 
certain  groups  of  bacteria  and  molds. 

Ammoniuyn  salts  represent  fairly  good  sources  of  nitrogen  for  micro- 
organisms, and  are  superior  in  this  respect  to  nitrates.  The  assimila- 
tion  of  ammonia  by  soil  organisms  is  one  of  the  reasons  why  in  fertilizer 
tests  the  nitrogen  applied  as  ammonium  often  does  not  act  as  well  as 
does  nitrate  nitrogen. 

Free  nitrogen  is  least  suitable  and  can  be  assimilated  only  by  a com- 
paratively small  number  of  microorganisms.  But  even  these  prefer 
nitrates  and  ammonium  salts,  and  some  of  them  grow  still  more  vigor- 
ously in  the  presence  of  amides  and  proteins. 

Carbon  Requirement. — Again  practically  all  carbonaceous  com- 
pounds occurring  in  nature  may  serve  as  food  to  some  or  to  many  micro- 
organisms. 

Proteins  can  nearly  always  serve  simultaneously  as  sources  of  nitro- 
gen as  well  as  of  carbon.  But  some  species,  for  instance  certain  lactic 
acid  bacteria,  are  so  fastidious  that  they  will  grow  only  if  sugar  is  added 
to  their  protein  food,  as  naturally  occurs  in  milk. 

1 Kresling,  Centralbl.  f.  Bakt.,  I.  Abt.  Bd.  30,  1901,  p.  897. 


42 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


Among  the  carbohydrates  sugars  are  usually  better  than  starch,  and 
this  in  turn  is  generally  better  than  cellulose;  but  some  bacteria  prefer 
the  latter  very  much,  and  may  even  be  unable  to  grow  in  the  presence  of 
sugars. 

Certain  alcohols,  like  glycerol  and  mannitol,  are  also  widely  used,  as 
are  many  organic  acids  in  the  form  of  their  salts  (malates,  lactates, 
citrates,  succinates,  etc.). 

Carbon  dioxide,  the  main  source  of  carbon  for  the  higher  plants,  can 
serve  the  same  purpose  with  only  a few  groups  of  bacteria ; and  still 
smaller  is  the  number  of  microorganisms  capable  of  living  on  carbon 
monoxide  or  on  methane,  but  these  groups  are  of  considerable  importance 
in  the  regular  progress  of  carbon  transformations  in  nature. 

Mineral  Requirements. — The  same  elements  which  constitute  the 
inorganic  parts  of  the  higher  organisms,  especially  phosphorus,  potas- 
sium, sulfur,  iron,  calcium,  and  magnesium,  are  equally  necessary  for  all 
microorganisms.  Several  experiments  have  been  made  which  seemed  to 
indicate  that  some  of  these,  namely  sulfur,  potassium,  calcium,  and  iron, 
were  not  essential.  But  it  is  next  to  impossible  to  exclude  the  last  traces 
of  these  elements  from  vessels  and  substrates  used  in  such  experiments,1 
and  a weak  growth  of  bacteria  (with  about  0.7  per  cent  total  minerals 
in  the  fresh  substance),  weighing  only  a few  thousandths  of  a milligram, 
needs  such  infinitesimal  quantities  of  these  elements  that  the  results  ob- 
tained are  never  fully  convincing.  It  is  certain  that  in  the  presence  of 
these  elements  a better  growth  was  always  observed.  Especially  in  regard 
to  phosphorus  and  calcium  it  has  been  noticed  repeatedly  that  the  bac- 
terial requirements  are  sometimes  distinctly  greater  than  those  of  the 
higher  plants  ;2  therefore,  in  addition  to  the  direct  effect  on  the  crops 
the  application  of  calcium  phosphates  may  increase  the  activity  of  nitri- 
fying and  nitrogen  fixing  bacteria  in  the  soil,  and  thus  prove  helpful  in 
an  indirect  way. 

Other  mineral  elements,  especially  aluminum,  manganese,  and  silicon, 
have  been  also  found  distinctly  beneficial  in  some  cases.3  They  are 
usually  classed  as  stimulants  and  will  be  discussed  as  such  below. 

Total  Food  Supply. — The  various  inorganic  as  well  as  organic 
nutrients  may  be  more  or  less  readily  used  according  to  the  combination 
in  which  they  are  offered.  Sodium  may  replace  part  of  the  potassium,  a 
rich  carbonaceous  food  supply  may  increase  the  availability  of  a poor 

1 Details  are  given  in  a paper  by  W.  Benecke,  Botan.  Ztg.  54,  1896,  I.  Abt.,  p.  97 ; 
65,  1907,  I.  Abt.,  p.  1. 

2 Lohnis,  “Handbuch  der  landwirtschaftlichen  Bakteriologie,’’  1910,  p.  75S. 

3 H.  Kaserer,  Zeitschr.  f.  d.  landw.  Versuchswesen  in  Oesterreich,  Bd.  14,  1911. 
p.  97. 


Plate  IV 


1.  Development  of  Bacteria  in  Tapwater  + 0.05%  K2HP04 
+ 0.5%  Peptone,  0.5%  Asparagin,  0.5%  Ammonium  Sulfate,  0.5%  Nitrate 


2.  Growth  of  Bacteria  and  Fungi  in  Tapwater  + 0.05%  K2HP04 
+ 1%  Glycerol  and 

0.5%  Peptone,  0.5%  Asparagin,  0.5%  Ammonium  Sulfate,  0.5%  Nitrate 


(Facing  page  ^2) 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  43 


nitrogen  source,  and  vice  versa.  The  culture  flasks  shown  in  Plate  IV 
illustrate  this  fact.  All  of  them  contained  tap  water  with  0.05  per  cent 
dipotassium-phosphate,  to  which  per  cent  peptone,  asparagin, 
ammonium  sulfate,  or  nitrate  had  been  added.  Those  in  the  lower  row 
received  in  addition  1 per  cent  glycerol ; small  amounts  of  soil  were  used 
for  inoculation  in  all  cases.  In  both  rows  the  peptone  and  asparagin  solu- 
tions show  good  growth  of  whitish  masses  of  bacteria,  while  the  ammonium 
sulfate  and  the  nitrate  solutions  without  glycerol  remained  perfectly 
clear.  In  presence  of  glycerol,  however,  the  development  is  equal 
to  that  in  the  other  flasks.  Some  floating  mold  colonies  are  visible  on 
the  ammonium-sulfate  glycerol  solution. 

Bacteria  and  fungi  must  have  their  food  in  soluble  forms,  while 
protozoa  can  devour  solid  food.  Very  different  solvents,  so-called 
enzymes,1  are  produced  by  bacteria  and  fungi  in  order  to  make  sub- 
stances accessible  to  them  which  are  as  such  insoluble  in  water.  Starch, 
cellulose,  fat,  and  many  other  nutrients  belong  to  this  class. 

The  minimum  quantities  of  food  which  still  allow  bacterial  growth, 
are  extremely  small.  Distilled  water,  kept  in  the  laboratory  for  some 
time,  often  becomes  rich  in  bacteria  and  molds.  Exact  tests  of  the  food 
requirements  of  water  bacteria2  have  shown  that  these  organisms  are  suffi- 
ciently supplied  if  they  find  in  1000  cc.  water : 0.002  yy  dextrose,  and 
0.00007  yy  ammonium  sulfate  (1^7=1/1000  mg.)  ; or  1 part  dextrose  in 
500,000  million  parts  of  water,  and  1 part  ammonium  sulfate  in  14,000,- 
000  million  parts  of  water.  These  almost  incredibly  small  figures  be- 
come intelligible  by  the  following  consideration.  If  1 cc.  water  contains 
1000  bacterial  cells,  there  are  1 million  of  them  in  1000  cc.,  which  have 
a weight  of  approximately  1 yy  and,  accordingly,  need  0.002  of  their 
own  weight  in  sugar,  and  0.00007  of  it  in  ammonium  sulfate.  A man  is 
sufficiently  supported  if  he  eats  daily  of  carbohydrates  0.005,  and  of 
proteins  0.0007  of  his  own  weight,  that  is,  comparatively  not  much  more 
than  those  bacteria  need. 

Stimulants. — It  was  mentioned  above  that  certain  substances,  which 
exert  a distinctly  favorable  influence  when  present  in  very  small 
amounts,  are  usually  classed  as  stimulants,  not  as  food.  That  is,  for  in- 
stance, the  case  with  manganese.  A French  author,  G.  Bertrand  3 pointed 
out  that  if  only  1 mg.  of  this  element  was  added  to  10  million  cc.  of 
nutrient  solution,  the  “stimulating”  effect  became  quite  noticeable. 
However,  this  quantity  is  equivalent  to  0.1  yy  per  1000  cc. ; in  other 

1 Derived  from  ffyoj  (zyme)  = ferment. 

2 E.  Kohn,  Centralbl.  f.  Bakt..  II.  Abt.  15,  1906,  pp.  690,  777. 

3 G.  Bertrand,  Comyt.  rend.  Acad.  Paris,  tome  154,  1912,  p.  616. 


44 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


words,  the  amount  of  manganese  was  much  larger  than  that  of  sugar 
and  ammonium  salt  in  the  example  just  discussed.  Many  organic  and  in- 
organic substances  have  been  classed  as  stimulants  which  may,  in  fact, 
just  as  well  be  considered  as  accessory  food  elements.  That  many  of 
them  exert  a poisoning  influence  when  present  in  large  quantities  would 
also  not  militate  against  this  view.  It  is  worth  noting  that  those  mineral 
salts  (sodium  salts  and  borates)  which  contribute  to  the  sterility  of  part 
of  the  alkali  soils,  act  in  an  analogous  manner,  viz.,  favorably  in  small, 
unfavorably  in  large  quantities.1 

Influence  of  Reaction. — Bacteria  prefer  generally  a neutral  or 
slightly  alkaline,  yeasts  and  molds  a slightly  acid  reaction.  But  this  rule 
again  has  its  exceptions.  Lactic,  acetic,  sulfuric,  and  other  acids  are 
produced  by  bacteria  which  can  withstand  a rather  high  degree  of 
acidity.  But  if  raw  meat  is  covered  with  milk  in  which  acid  forming 
bacteria  predominate,  it  is  protected  against  the  attacks  of  putrefying 
bacteria.  The  protein  substances  in  cheese  are  equally  protected  from 
putrefaction  by  the  lactic  acid  first  formed.  It  is  well  known  that  accu- 
rate control  of  the  acidity  of  milk,  cream,  and  rennet  infusions  is  of 
great  importance  for  producing  butter  and  cheese  of  high  quality. 
Practical  experience  has  discovered  that  such  a control  is  the  best  way 
to  regulate  the  ripening  processes,  and  bacteriology  has  furnished  the 
explanation  and  also  a more  definite  knowledge  of  these  facts. 

By  their  own  activity  bacteria  and  molds  may  frequently  change  the 
initial  reaction.  Milk,  at  first  nearly  neutral,  becomes  distinctly  acid  by 
the  action  of  bacteria,  then  molds  begin  to  grow  on  the  surface,  con- 
suming the  acid  and  producing  alkali.  If  kept  for  a long  time  a distinctly 
alkaline,  brownish  liquid  of  offensive  odor  results.  In  soil,  where  large 
amounts  of  proteins  are  absent,  the  tendency  to  acid  formation  usually 
prevails ; accordingly  liming  becomes  necessary  from  time  to  time  to 
neutralize  these  acids  or,  as  it  is  sometimes  called,  to  “sweeten”  the 
ground.  In  other  cases  increased  acidity  may  prove  helpful  as  a means 
to  suppress  the  growth  of  certain  detrimental  organisms,  for  instance  of 
those  causing  “scab”  of  potatoes.2 

2.  PHYSICAL  FACTORS 

While  in  most  cases  the  food  requirements  of  higher  plants  and 
animals  are  distinctly  specialized,  many  microorganisms  are  known  to  be 
able  to  adapt  themselves  to  more  or  less  different  kinds  of  nutrition.  In 

1 C.  B.  Lipman,  Centralbl.  f.  Bakt.,  II.  Abt.  Bd.  32,  1911,  p.  58;  Bd.  33,  1912,  p.  305; 
Bd.  41,  1914,  p.  430. 

2 W.  H.  Martin,  Soil  Science,  vol.  IX,  1920,  p.  393,  XI,  1921,  p.  75. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  45 


the  same  manner  their  relations  to  various  physical  factors,  as  moisture, 
air,  light,  etc.,  are  much  more  varied  than  those  of  the  higher  organisms. 
Practically  every  chemical  substance  occurring  in  nature  can  serve  as 
food  for  one  or  another  group  of  microorganisms ; and  even  under  most 
extreme  physical  conditions,  where  higher  plants  and  animals  can  not 
exist,  bacteria  still  continue  to  live  and  to  perform  ceaselessly  their  work 
of  transformation  and  destruction. 

Water  Requirement. — Vegetative  bacterial  cells  contain  75  to  98 
per  cent  water.  Accordingly,  bacterial  life  and  activity  are  not  possible 
without  sufficient  moisture.  Protozoa  need  still  more;  they  are  true 
water  organisms.  Molds,  on  the  other  hand,  prefer  relatively  dry  sub- 
strates, as  is  indicated  by  their  growth  on  stale  bread,  old  leather,  etc. 

Frequently  it  depends  solely  on  the  water  content  of  a substance 
whether  it  will  be  attacked  by  molds  or  by  bacteria.  Comparatively  dry 
stable  manure  becomes  moldy,  but  when  kept  moist,  typical  putrefaction, 
due  to  bacterial  action,  takes  place.  Dry  parts  of  silage  show  mold 
growth,  as  does  damp  hay.  If  the  water  content  of  grain,  roughage,  or 
of  concentrated  animal  feed  (for  instance,  in  oil  cakes  and  cottonseed 
meal)  is  below  12  per  cent,  they  are  protected  against  both  molds  and 
bacteria.  If  more  water  is  present,  molds  begin  to  appear,  which  by  their 
respiration  produce  carbon  dioxide  and  water;  the  latter  accumulates  and 
increases  gradually  the  original  amount  until — if  time  permits — enough 
water  becomes  available  for  bacterial  growth  and  for  complete  spoilage 
of  the  fodder.  Materials  containing  fat  are  especially  liable  to  underge 
such  alterations.  For  instance,  powdered  rape  cake  was  kept  for  two 
years  and  showed  the  following  composition  d 


Percentage 

Fat 

Water 

At  the  beginning 

Sample  I 
10.53 
1.98 

Sample  II 
8.50 
1.87 

Sample  I 
12.45 
21.94 

Sample  II 
12.31 
23.42 

At  the  end 

Normally  such  material  will  be  used  before  decomposition  can  go  so  far. 
But  this  example  explains  why  a moldy  odor  and  even  visible  mold 
growth  are  so  common  on  all  foodstuffs  not  kept  perfectly  dry. 

Soil  Moisture. — Soil  with  10  to  12  per  cent  water  appears  fairly  or 
completely  dry  to  sight  and  to  touch ; yet  this  amount  of  moisture  is 
usually  sufficient  for  undisturbed  bacterial  activity.  Different  soils 

1 H.  Ritthausen  und  Baumann,  Landwirtschaftliche  Versuchsstationen,  Bd.  47, 1896, 
p.  389. 


46 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


behave  differently,  and  it  was  reported  in  the  French  literature,  for 
instance,  that  in  one  soil  nitrification  took  place,  although  its  water  con- 
tent was  as  low  as  7.3  per  cent,  while  in  another  soil  this  bacterial  proc- 
ess required  more  than  16.5  per  cent.1  Similar  apparently  contra- 
dictory statements  are  rather  frequent  in  the  literature,  but  they  are 
due  merely  to  incorrect  methods  of  recording  the  facts.  The  amount  of 
water  present  in  a soil  should  not  be  given  in  percentage  of  the  weight 
of  the  soil,  but  in  percentage  of  its  water-holding  capacity.  This  is 
low  in  sand  (usually  10  to  15  per  cent  of  the  soil  weight),  high  in  loam 
and  clay  (about  20  to  30  per  cent),  and  still  higher  in  peat  (40  to  60 
per  cent  and  more).  A saturation  of  60  to  80  per  cent  of  the  total  water- 
holding capacity  of  a soil  represents  the  optimum  moisture  for  plant 
growth  as  well  as  for  normal  bacterial  action.2  If  less  than  one  half  of 
the  water-holding  capacity  is  saturated,  plants  and  bacteria  will  gradually 
dry  out,  but  if  the  water  content  is  very  high,  only  certain  plants 
and  bacteria  together  with  the  protozoa  will  persist.  Acids  and  other 
noxious  products  accumulate;  the  soil  becomes  sour,  swampy,  and  use- 
less for  most  agricultural  purposes  if  not  drained. 

Effect  of  Concentration. — Since  the  bacteria  do  not  live  in  chem- 
ically pure  water,  but  in  more  or  less  concentrated  solutions  of  various 
substances,  the  effect  exerted  by  high  concentrations  sometimes  becomes 
of  great  importance.  In  fact,  adding  large  amounts  of  sugar  or  salt  to 
substances  of  high  water  content  is  a very  old  and  widely  used  means  of 
protecting  such  materials  against  bacterial  invasion.  Highly  sweetened 
fruit  jams,  jellies,  and  molasses  contain  so  little  available  moisture  that 
this  will  suffice  only  for  a moderate  growth  of  molds.  Sweetened  con- 
densed milk  may  still  contain  numerous  living  bacteria,  but  they  remain 
inactive  on  account  of  the  high  concentration.  Salted  meat  and  fish 
present  other  examples  of  this  kind,  although  the  sodium  chloride  itself 
exerts  its  additional  detrimental  effect,  as  it  does  in  water  or  in  soil  of 
high  salt  content.  Some  bacteria  and  fungi,  however,  can  endure  the 
strong  osmotic  pressure  exerted  by  very  large  amounts  of  soluble  sub- 
stances. One  species  {Bad.  vernicosum),  once  found  in  cotton  seed 
meal,  was  not  seriously  harmed,  for  instance,  by  the  following  concen- 
trations :3 

Saccharose  70  per  cent,  Lactose  50  per  cent,  Sodium  chloride  18-20  per  cent, 
Dextrose  70  per  cent,  Glycerin  40  per  cent,  Magnesium  sulfate  25-28  per  cent. 

1 Deherain,  Com-pt.  rend.  Acad.  Paris,  tome  125,  1897,  p.  282. 

2 Lohnis,  “Handbuch  der  landwirtschaftlichen  Bakteriologie,”  1910,  p.  737. 

3 Zopf,  Beitrdge  zur  Physiologie  und  Morphologie  niederer  Organismen,  Bd.  1,  1892, 

p.  80. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  47 


Effect  of  Drought. — If  lack  of  available  water  would  end  bacterial 
life  as  quickly  as  it  terminates  the  existence  of  higher  plants  and  animals, 
the  metabolic  processes  caused  by  bacteria  in  the  soils  could  not  occur 
with  such  regularity  as  they  do.  Bacteria  and  fungi,  as  well  as  protozoa, 
are  able  to  produce  resting  forms  which  show  an  increased,  sometimes 
even  a surprisingly  high  resistance  against  drought  and  other  harmful 
influences.  Nor  are  the  vegetative  cells  easily  killed  by  lack  of  water. 
The  slime  or  the  capsules  which  surround  most  of  these  minute 
cells  afford  considerable  protection.  Furthermore,  soil  which  is  to  all  ap- 
pearances completely  dry,  still  holds  small  amounts  of  water  in  cracks 
and  holes  of  its  inorganic  and  organic  constituents,  where  numerous 
bacteria  may  find  a temporary  refuge. 

If  bacteria  are  dried  in  very  thin  layers,  as  in  the  dust  of  rooms  and 
on  the  streets,  the  death  rate  among  the  vegetative  cells  is  comparatively 
high.  Repeated  changes  from  wet  to  dry  act  also  in  a very  unfavorable 
manner.  Quick  drying  in  vacuo,  on  the  other  hand,  tends  to  keep  cul- 
tures alive.  But  no  noticeable  activity  is  to  be  expected,  of  course,  under 
such  conditions.  Rapid  drying  and  storage  in  dry  rooms  is,  therefore, 
widely  used  to  protect  human  and  animal  foodstuffs  cheaply  and  success- 
fully from  the  attacks  of  all  kinds  of  microorganisms. 

Oxygen  Requirement;  Respiration. — All  higher  organisms  need  the 
free  oxygen  of  the  air  for  respiration,  that  is,  for  the  internal  combus- 
tion of  organic  substances,  by  which  means  warmth  and  energy  are  pro- 
duced. Many  bacteria,  fungi,  and  protozoa  breathe  in  the  same  manner, 
but  certain  groups  behave  differently.  Some  oxidize  inorganic  instead  of, 
or  as  well  as,  organic  substances.  Others  can  live  in  the  presence  as  well 
as  in  the  absence  of  air ; and  still  another  group  is  directly  poisoned  by 
free  oxygen ; it  can  develop  only  in  the  absence  of  air,  although  a slow 
and  gradual  adaptation  to  the  normal  mode  of  respiration  has  been 
brought  about  with  some  of  these  organisms. 

Figure  16  shows  three  cylinders  partly  filled  with  nutrient  gelatin, 
each  of  which  had  been  inoculated  with  different  kinds  of  bacteria.  Ac- 
cording to  their  relation  to  the  oxygen  of  the  air,  three  characteristic 
types  of  so-called  stab  cultures  have  developed.  In  cylinder  A some 
whitish  growth  is  visible  on  and  in  the  upper  part  of  the  gelatin,  in  B 
the  growth  along  the  needle  track  reaches  from  top  to  bottom,  but  in  C 
some  cloudy,  flocculent  development  took  place  only  in  the  lower  third  of 
the  substrate. 

Anaerobic  Life. — Pasteur,  who  made  the  first  thorough  studies  upon 
these  different  behaviors  of  the  bacteria,  named  the  two  groups  living  re- 
spectively in  the  presence  and  in  the  absence  of  air,  “aerobies  et 


48 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


anaerobies.  ’ n Since  then  it  has  become  customary  to  define  them  as  strictly 
aerobic,  or  strictly  anaerobic,  while  the  intermediate  group  is  usually 
termed  facultative  anaerobic  or  facultative  aerobic.  Beijerinck2  is  of  the 
opinion  that  the  anaerobic  organisms  also  need  a small  amount  of  free  oxy- 
gen, and  calls  them  therefore  microaerophilie.  But  an  exact  proof  of  the 


a be 

Fig.  16. — Gelatine  stab  cultures  (■§•  nat.  size),  (a)  aerobic,  ( b ) facultative  anaerobic, 
(c)  strictly  anaerobic  growth. 

correctness  of  this  assumption  is  lacking,  and  it  is  more  probable  that 
the  contrary  view  holds  true,3  although  a final  decision  is  practically  im- 

1 Compt.  rend.  Acad.  Paris,  tome  56,  1863,  p.  1192,  note  1.  The  terms  are  derived 
from  a-f)p  (aer)  =air,  and  £/os  (bios)  = life. 

2 Arch,  neerland.,  2 ser.,  vol.  2,  1899,  p.  397. 

3 Burri  und  Kursteiner,  Centralbl.  f.  Bakt.,  II.  Abt.  Bd.  21.  190S,  p.  2S9. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  49 


possible.,  because  minute  traces,  hardly  detectable  even  by  the  most  re- 
fined chemical  methods,  may  still  mean  much  to  the  equally  minute  bac- 
teria, as  was  discussed  in  the  preceding  chapter. 

Anaerobic  bacteria  play  a very  important  role  in  nature.  If  they 
were  absent,  all  I’esidues,  corpses,  etc.,  buried  deep  in  water-logged 
ground,  would  not  be  decomposed  at  all,  but  preserved  for  centuries. 
On  the  surface  of  the  ground,  too,  the  destructive  processes  would  take 
much  more  time,  if  dead  bodies,  for  instance,  were  attacked  only  from 
the  outside  by  aerobic  organisms.  But  by  the  combined  “front  and 
rear  attacks”  of  aerobic  and  anaerobic  bacteria  surprisingly  rapid  de- 
structions are  secured. 

Oxygen  Maxima  and  Minima. — Special  investigations  upon  the 
limits  of  oxygen  pressure,  endurable  to  aerobic,  facultative  anaerobic, 
and  to  strictly  anaerobic  bacteria,  respectively,  have  shown1  that  rep- 
resentatives of  the  last  named  group  are  unable  to  grow  in  an  atmos- 
phere containing  more  than  0.13  to  1.04  per  cent  free  oxygen,  which  is 
less  than  1/100  to  1/10  of  the  volume  usually  present  in  air.  Facultative 
as  well  as  strictly  aerobic  bacteria  have  in  most  cases  very  low  oxygen 
minima  (usually  close  to  0),  while  their  maxima  were  found  to  be  be- 
tween 2 and  6,  usually  around  3 to  4 atmospheres.2  Such  wide  ranges 
of  endurable  oxygen  concentration  are  again  quite  exceptional,  but  this 
fact  explains  why  increased  pressure  of  common  air  has  hardly  any 
detrimental  effect  upon  aerobic  and  facultative  anaerobic  bacteria.  Even 
a quick  increase  to  3000  atmospheres  pressure  proved  to  be  of  very 
little  effect,  which,  however,  became  quite  noticeable  if  sudden  changes  of 
high  and  low  pressure  were  several  times  repeated.3  Pressures  of  7000 
atmospheres  killed  vegetative  cells  of  bacteria  and  fungi  after  a few 
minutes.4 

Oxidation  and  Reduction. — Anaerobic  as  well  as  aerobic  life  de- 
pends, of  course,  on  the  continuous  oxidation  caused  by  respiration. 
But  while  free  oxygen  from  the  air  is  used  by  the  aerobes  for  this  pur- 
pose, only  compounds  rich  in  oxygen,  as  sugar,  nitrates,  sulfates,  etc., 
are  used  by  the  strictly  anaerobic  organisms.  The  fixed  oxygen  is  taken 
from  these  substances,  and  they  undergo  therefore  more  or  less  far- 
reaching  reductions  to  methane  and  hydrogen,  nitrites  and  ammonia,  sul- 
fites and  sulfides.  Because  nitrate  contains  relatively  more  oxygen  than 
does  sugar,  it  is  used  to  protect  the  latter  against  the  attacks  of  facultative 

1 Chttdiakow,  Centralbl.  f.  Bakt.  II.  Abt.  Bd.  4,  1898,  p.  389. 

2 Porodko,  Jahrbucherf.  wissenschaftliche  Botanik,  Bd.  41,  1904,  p.  61. 

3 Chlopin  und  Tammann,  Zeitschr.  f.  Hygiene,  Bd.  45,  1903,  p.  171. 

4 Hite,  Giddings  and  Weakley,  W.  Va.  Agr.  Exp.  Stat.  Bull.  146,  1914. 


50 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


and  strictly  anaerobic  bacteria  in  cheese,  where  in  the  absence  of  nitrate 
the  milk  sugar  might  be  reduced  under  liberation  of  hydrogen,  resulting 
in  gassiness  of  the  cheese. 

There  are  numerous  other  cases  where  according  to  circumstances  the 
activities  of  aerobic  or  of  anaerobic  organisms  are  favored  in  order  to 
attain  the  desired  oxidation  or  reduction.  Drainage,  tillage,  and  cultiva- 
tion of  the  soil  assure  a fairly  thorough  aeration  leading  to  the  oxidation 
of  organic  residues  and  to  the  formation  of  nitrates.  In  the  silo  and  in 
rotting  manure  all  practical  means  are  used  to  insure  the  exclusion  of  air 
and  thereby  the  activity  of  anaerobic  bacteria,  which  are  useful  in  these 
cases. 

Organic  and  Inorganic  Respiration. — Carbonaceous  organic  sub- 
stances, such  as  sugars,  fats,  and  acids,  are  the  sources  of  energy  in  the 
respiratory  processes  of  most  of  the  lower  as  well  as  of  all  higher  organ- 
isms. But  the  intensity  of  oxidation  is  again  comparatively  much  greater 
with  the  minute  bacteria,  because  of  their  relatively  large  active  surfaces. 
Comparative  tests  have  shown,  for  instance,  that  the  following  amounts 
of  carbon  dioxide,  calculated  in  percentage  of  the  weight  of  the  various 
organisms,  were  daily  produced  d 

By  beet  roots  .075  per  cent,  By  molds  2-2.7  per  cent, 

By  roots  of  cereals  1-2  per  cent,  By  bacteria  7-37 . 5 per  cent, 

The  intensive  carbon  dioxide  formation  steadily  going  on  in  silos,  in 
manure,  in  the  soil,  etc.,  is  of  great  importance  in  many  respects,  which 
will  be  discussed  later. 

The  energy  liberated  by  the  respiration  of  organic  substances  origi- 
nates from  the  sun,  and  is  accumulated  by  the  green  plants  producing 
these  substances.  All  organisms  living  in  this  manner,  and  they  are  by 
•far  in  the  majority,  are  in  fact  motors  driven  by  the  energy  of  sunshine. 
But  there  are  certain  groups  of  bacteria,  able  to  derive  their  energy  from 
the  oxidation  of  inorganic  substances,  and  therefore  to  live  independent 
of  solar  energy.  They  oxidize  ammonia,  or  hydrogen,  or  hydrogen  sul- 
fide, to  nitrous  and  nitric  acid,  to  water,  or  to  sulfuric  acid,  respectively. 
These  are  processes  of  great  physiological,  and  in  part  also  of  consider- 
able practical  importance.  Naturally,  the  oxidation  of  organic  com- 
pounds is  not  entirely  absent  in  these  organisms,  because  their  own  bodies 

1 These  calculations  are  based  on  data  published  by  Stoklasa  et  al.  in  Centralbl.  f. 
Bakt.,  II.  Abt.  Bd.  14,  1905,  p.  727;  Bd.  29,  1911,  pp.  401,  457;  Berichte  d.  Deuisch. 
Bolan.  Gesellschaft,  Bd.  24,  1906,  p.  22;  Jahrb.f.  unssensch.  Botanik,  Bd.  46,  190S,  pp.  73, 
80;  and  by  Bassalik  in  Zeitschr.  f.  Garungsphysioleaie,  Bd.  2,  1912,  p.  27.  The  results 
given  for  dry  weight  were  changed  to  figures  for  fresh  weight  by  assuming  that  beet 
roots  and  bacteria  contained  15  per  cent,  roots  of  cereals  30  per  cent  dry  substance. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  51 


are  built  up  of  such  substances.  The  main  source  of  energy,  however, 
which  also  enables  them  to  assimilate  carbonic  acid  and  to  produce  in 
complete  darkness  the  organic  compounds  which  they  need,  is  represented 
by  those  various  modes  of  ‘ ‘ inorganic  respiration.  ’ ’ 

Influence  of  Temperature. — The  vegetative  cells  of  microorganisms 
are  filled  with  a mixture  of  protein  substances  and  much  water.  Because 
the  latter  freezes  at  0°  C.  and  the  former  coagulate  at  70°  to  80°  C.,  active 
life  is  generally  limited  by  these  degrees.  Some  slow  action  at  tempera- 
tures below  0°  C.  is  occasionally  noticeable,  either  if  the  cell  solution  on 
account  of  its  concentration  does  not  freeze  promptly,  or  if  sufficient 
enzymes  are  present  to  continue  the  transformations  begun  at  higher  tem- 
peratures. The  gradual  deterioration  of  cold  storage  butter  illustrates 
these  principles.  At  very  low  degrees,  however,  even  such  activities  end, 
and  occasionally  corpses  of  mammoths  have  been  dug  out  from  solid 
ice  in  northern  latitudes,  so  completely  preserved  during  thousands  of 
years  that  their  meat  was  still  readily  eaten  by  the  dog  teams  of  the  ex- 
ploring parties. 

Slow  cooling  and  slow  freezing  do  not  kill  the  microorganisms  as  a 
rule.  Repeated  quick  alternations  of  freezing  and  thawing,  however, 
prove  to  be  detrimental  to  lower  as  well  as  to  higher  organisms.  Vegeta- 
tive cells  of  bacteria  and  fungi  have  been  kept  in  liquid  air,  and  even 
in  liquid  hydrogen  (at— 252°  C.)  without  serious  harm,  provided  that 
the  changes  from  freezing  to  thawing  were  not  too  rapid.1  Arctic  soils 
contain  the  same  microflora  as  soils  of  warmer  climates.2  And  the 
freezing  of  milk  is  without  influence  upon  the  lactic  acid  bacteria  living 
therein.3 

Bacterial  Life  at  Low  and  at  High  Temperatures. — Certain  micro- 
organisms prefer  low,  others  high  temperatures.  The  former  are  called 
psychrophilic  or  cryophilic  (cold  loving),  the  latter  thermophilic 
(warmth  loving). 

Psychrophilic  bacteria  are  common  in  water  of  low  temperature, 
they  predominate  in  soil  during  the  cold  season,  and  they  multiply 
quickly  in  milk  and  butter  kept  at  low  temperature.  An  increase  from 
2 to  4500  millions  per  cc.  was  observed,  for  instance,  in  milk  kept  4 weeks 
at  approximately  0°  C.4  Typical  lactic  acid  bacteria  work  very  slowly 
below  10°  C. ; therefore  a more  or  less  abnormal  microflora  develops  in 
milk  kept  for  some  time  at  low  degrees,  which  causes  a deterioration 

1 A.  Macfadyean  and  S.  Rowland,  Proc.  Roy.  Soc.  (London),  vol.  66, 1900,  p;p,  339, 
448;  vol.  71,  1902,  p.  76. 

2 Barthel,  Meddel.  om  Gronland,  LXIV,  1922. 

3 B^dinow,  Centralbl.  f.  Bakt.,  II.  Abt.,  Bd.  34,  1912,  p.  183. 

4 Lohnis,  ‘‘Handbuch  der  landwjrtschaftlichen  Bakteriologie,”  1910,  p.  150. 


52 


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similar  to  that  noticeable  in  pasteurized  milk  kept  some  days  at  a higher 
temperature.  The  characteristic  disagreeable  flavor  often  noticeable  in 
food  kept  a few  days  in  the  ice  box  is  also  partly  caused  by  such  psychro- 
philic  organisms. 

In  a moderate  climate  those  species  predominate  in  nature  which 
have  their  temperature  minimum  close  to  0°  C.,  their  optimum  at  10°  to 
20°  to  30°  C.,  and  their  maximum  in  the  neighborhood  of  40°  C.  Un- 
favorable conditions,  lack  of  food,  etc.,  narrow  the  range  of  suitable  tem- 
peratures, while  it  may  expand  more  or  less  if  other  circumstances  are 
very  favorable. 

Thermophilic  bacteria  have  their  minimum  at  approximately  35°  C., 
their  optimum  at  50  to  65°  C.,  and  their  maximum  at  75  to  80°  C.  They 
are  numerous  in  soils  of  hot  climates,  in  hot  wells,  and  in  loosely  packed 
moist  organic  substances  where  temperatures  rise  quickly,  sometimes  to 
the  point  of  ignition  (as  in  damp  hay,  cotton,  horse  manure,  etc.).  The 
thermophilic  bacteria  themselves  participate  by  their  respiration  in  the 
production  of  heat,  and  are  in  turn  stimulated  by  the  rising  temperature 
until  the  maximum  is  reached.  In  regard  to  their  active  participation 
they  are  called  thermogenic  (heat  producing). 

Influence  of  Light, — It  is  a well-known  fact  that  sunlight  helps  to 
prevent  and  to  drive  out  diseases.  Contrary  to  the  green  plants,  which 
can  not  live  without  light,  because  the  chlorophyll  action  depends  on  it, 
most  of  the  bacteria  and  fungi  are  hurt  or  even  killed  by  direct  sunlight. 
Especially  the  disease  producing  germs  are  very  sensitive,  while  other 
groups  are  more  resistant.  The  majority  of  soil,  manure,  and  milk  bac- 
teria are  practically  unaffected  by  diffuse  daylight,  but  intensive  sun- 
shine reduces  their  numbers  quickly  if  thin  layers  are  exposed.  For  in- 
stance, only  1/5  or  1/6  of  the  bacteria  originally  present  in  a soil  sample 
survived  when  this  was  exposed  to  bright  sunshine  for  five  hours  in  a 
layer  only  1 mm.  deep.1  In  thin  layers  of  gravel  moistened  by  dilute 
urine  the  following  amounts  of  nitrogen  were  transformed  into  nitrate  :2 


Sample  I 

Sample  II 

In  the  light 

19  mg. 

110  mg. 

In  darkness . . . . . 

360  mg. 

In  a few  cases  sunlight  acts  favorably  by  stimulating  the  pigmenta- 
tion of  certain  bacteria,  and  in  soil  as  well  as  in  water  the  growth  of  algae 
is  naturally  increased.  A good  growth  of  soil  algae  means  a better  sup- 
ply of  organic  substances  for  the  soil  bacteria,  especially  for  those  fixing 

1 Kedzior,  Archivf.  Hyg.,  Bd.  36,  1S99,  p.  323. 

s Soyka,  Zeitsch.  f.  Biologie,  Bd.  14,  1878,  p.  466. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  53 


nitrogen  from  the  air.  In  water  the  purifying  action  of  algae  is  added 
to  that  of  the  direct  bactericidal  effect  of  sunlight. 

The  real  causes  of  the  detrimental  effect  of  light  are  not  yet  fully 
known ; but  it  is  certain  that  they  are  not  always  the  same.  Physical  as 
well  as  chemical  actions  may  participate;  heating,  drying,  formation  of 
peroxide  of  hydrogen  and  of  acids  in  organic  substrates  are  some  of 
these  possibilities.  Furthermore,  the  living  protoplasm  within  the  bac- 
terial cell  is  also  directly  affected  by  strong  light. 

Effects  of  Other  Physical  Factors. — Ultraviolet  rays  are  still  more 
detrimental  than  is  sunlight ; successful  application  of  this  fact  is  made  in 
water  purification  (see  Chapter  VI).  X-rays,  on  the  other  hand,  have 
very  little  or  no  effect.1  Radium  rays  are  more  or  less  harmful ; bacteria 
so  treated  become  radioactive  themselves.2  Electric  currents  may  act 
favorably  or  unfavorably,  according  to  circumstances.  Physical  as  well 
as  chemical  reactions  take  place ; these,  not  the  electricity  as  such,  are 
responsible  for  the  results  obtained.3 

Mechanical  treatment  may  also  cause  widely  varying  effects.  Vigor- 
ous shaking,  especially  in  the  presence  of  hard  bodies  (small  glass  beads, 
etc.),  naturally  destroys  the  soft  cells  of  bacteria,  but  reproductive  or- 
gans, like  the  minute  gonidia,  may  survive  and  later  produce  new  vegeta- 
tive growth.  Less  severe  shaking  may  merely  separate  the  cells  previously 
united  in  colonies,  and  thereby  stimulate  their  multiplication.  At  the 
same  time  a more  thorough  mixing  with  the  substrate  and  increased 
aeration  may  lead  to  more  vigorous  chemical  action.  For  instance,  soil 
samples,  after  being  kept  in  pots  for  several  weeks,  were  either  left 
untouched,  or  were  repeatedly  stirred  during  six  weeks,  and  were  then 
tested  with  regard  to  nitrification.  The  following  quantities  of  nitrates 
were  found  in  every  100  g.  of  soil  :4 

Sample  I Sample  II  Sample  III 
Untouched ....  2-3  mg.  2 mg.  2 mg. 

Stirred 39-44  mg.  46-51  mg.  57-71  mg. 

Similar  though  less  far-reaching  stimulating  effects  are  secured  by  thor- 
ough cultivation  of  the  fields,  or  by  emptying  and  refilling  of  pots  or 
pails  in  greenhouses. 

A special  influence  of  gravitation,  the  so-called  geotropism,  was  for- 

1 Beck  und  Schultz,  Archiv.  f.  Hyg.,  Bd.  23,  1896,  p.  495;  Wittlin,  Cenlralbl.  f. 
Bakt.,  II.  Abt.,  Bd.,  2,  1896,  p.  676. 

2 W.  Hoffmann,  Hygienische  Rundschau,  Bd.  13,  1903,  p.  913;  A.  R.  Green, 
Proc.  Roy.  Soc.  (London),  vol.  73,  1904,  p.  375. 

3 Lehmann  und  Zierler,  Archiv  f.  Hyg.,  Bd.  46,  1903,  p.  221. 

4 Deherain,  Compt.  rend.  Acad.  Paris,  tome  116,  1893,  p.  1094. 


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merly  held  responsible  for  a particular  type  of  growth  noticeable  with 
stab  cultures  of  certain  species  ( Proteus , Bac.  mycoides,  etc.)  when  grown 
in  gelatin.  From  the  needle  track  fine  branches  are  spreading  either  hori- 
zontally or  in  an  upward  direction.  This  was  explained  as  due  to  “nega- 
tive geotropism.  ’ ’ But  renewed  tests  have  shown  that  it  is  the  elasticity 
of  the  gelatine  which  decides  the  length  and  direction  of  these  lateral 
branches.  The  term  ‘ ‘ elasticotropism  ’ ’ was  therefore  proposed.1 

3.  SYMBIOSIS  AND  ANTAGONISM 

The  majority  of  microbiological  studies  is  made  with  pure  cultures  in 
the  laboratory.  Practically  all  our  present  knowledge  rests  on  such  in- 
vestigations. Yet  despite  the  very  great  value  of  this  kind  of  work,  it 
must  be  emphasized  that  much  remains  to  be  done  before  the  activities 
of  the  microorganisms  in  nature  are  fully  understood  and  explained. 
Associations  of  many  different  species  are  nearly  always  at  work  under 
natural  conditions,  and  the  environmental  conditions  themselves  are 
more  or  less  different  from  those  which  can  be  duplicated  in  the  labora- 
tory. If  only  experiments  with  pure  cultures  are  made  “our  conclusions 
might  be” — according  to  a very  proper  remark  made  by  Dr.  Chas.  E. 
Marshall2 — ‘ ‘ like  studying  man  apart  from  society  in  order  to  obtain  his 
social  relations.”  But  as  the  behaviors  and  activities  of  microorganisms 
show  much  more  variation  and  differentiation  than  is  known  among 
higher  plants  and  animals,  there  are  also,  of  course,  many  more  possibili- 
ties of  helpful  cooperation  or  of  vigorous  competition  in  the  struggle  for 
life. 

Symbiosis. — An  especially  interesting  example  of  mutually  helpful 
cooperation,  usually  called  symbiosis,3  is  furnished  by  the  leguminous 
plants  and  the  bacteria  living  in  their  root-nodules.  The  former  supply 
the  latter  with  large  quantities  of  carbohydrates,  and  receive  in  turn  all 
the  nitrogen  they  need  even  in  a soil  devoid  of  nitrogen  compounds,  and 
both  symbionts  are  evidently  greatly  benefited  by  this  exchange.  Prac- 
tically the  same  correlation  may  become  active  between  green  algae  and 
nitrogen  fixing  bacteria  in  water  as  well  as  in  soil.  In  water  especially 
so  much  organic  matter  can  be  produced  by  this  symbiotic  process  that 
a considerable  amount  of  fish  food  is  derived  from  this  source.4  Besides 
these,  there  are  numerous  other  possibilities  for  acquiring  a larger  amount 

1 H.  Zikes,  Centralbl.  f.  Bakt.,  II.  Abt.,  Bd.  11,  1903,  p.  59;  H.  C.  Jacobsen,  1.  c. 
Bd.  17, 1906,  p.  53;  H.  Kufferath,  Annales  de  VInstitut  Pasteur,  tome  25,  1911,  p.  601. 

2 Centralbl.  f.  Bakt.,  II.  Abt.,  Bd.,  11,  1904,  p.  740. 

3 The  Greek  word  (symbiosis)  means  “ living  together.” 

4 Hermann  Fischer,  Centralbl.  f.  Bakt.,  II.  Abt.,  Bd.  46,  1916,  pp.  304-320. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  55 


or  a more  suitable  kind  of  food  by  cooperative  action.  Of  special  interest 
in  this  respect  is  the  fact  that  new  bacterial  development  as  a rule 
proceeds  with  much  more  vigor  and  rapidity  when  it  starts  from  a con- 
siderable number  of  cells  of  the  same  kind,  not  from  a solitary  cell.  The 
stimulating  effect  of  such  an  association  is  also  noticeable  when  most  of 
these  cells  are  dead.  The  components  of  their  bodies  improve  the  food 
supply  of  the  surviving  cells. 

Alteration  of  the  reaction  of  the  substrates  plays  also  an  important 
role  in  symbiosis.  In  milk  and  in  cheese  the  activity  of  lactic  acid  bac- 
teria stimulates  the  growth  of  various  molds,  which  in  turn  destroy  the 
acids  formed  and  re-establish  an  alkaline  reaction,  thereby  favoring 
again  bacterial  growth.  Many  symbiotic  actions  contribute  to  the  ripen- 
ing of  cheese,  and  it  has  been  noticed  repeatedly  in  the  course  of  such 
experiments  that  pure  cultures  which  remained  more  or  less  inactive 
when  tested  separately,  developed  a vigorous  and  characteristic  action  if 
properly  mixed. 

Of  exceptional  importance  is  the  symbiosis  of  aerobic  and  anaerobic 
bacteria  in  nature.  The  extreme  sensitiveness  of  anaerobes  against  free 
oxygen  would  preclude  their  existence  and  activity  nearly  everywhere, 
if  it  were  not  that  the  presence  of  aerobic  bacteria  affords  protection. 
The  intensive  respiration  of  the  aerobes  removes  the  oxygen  from  the 
air  in  the  immediate  neighborhood,  replacing  it  by  carbon  dioxide ; other 
metabolic  products  may  add  to  the  beneficial  effect  of  this  symbiotic 
action.  After  numerous  anaerobic  cells  have  grown  they  themselves  are 
able  to  protect  the  younger  cells  in  an  analogous  manner.  Well  developed 
cultures  of  anaerobes  are  therefore  much  less  sensitive  against  free 
oxygen  than  are  young  ones  with  only  a small  number  of  cells.1 

Antagonism. — Besides  co-operation,  keen  competition  often  takes 
place  between  various  groups  of  microorganisms.  The  struggle  for  ex- 
istence is  no  less  violent,  although  less  spectacular  than  among  the  higher 
organisms.  Suitable  food  is  eagerly  attacked  by  many  different  species, 
and  the  more  active  kinds  quickly  outgrow  the  weaker  ones.  Rod-like 
bacteria  are  as  a rule  superior  to  cocci  in  this  respect  because  of  their 
larger  working  surface.  Acid  or  ammonia  producing  species  suppress 
their  competitors  by  creating  strongly  acid  or  alkaline  reactions,  as  in 
silage,  milk,  vinegar,  or  liquid  manure,  respectively. 

Very  distinctly  antagonistic  actions  take  place  whenever  disease  pro- 
ducing germs  try  to  invade  higher  plants  and  animals.  Only  compara- 
tively few  bacteria  are  able  to  overcome  the  acid  reaction  of  plant  saps, 
and  therefore  fungi  are  more  often  the  cause  of  plant  diseases.  In  the 

1 Burri  und  Ivursteiner,  Centralbl.  f.  Bait.,  II.  Abt.,  Bd.  21,  1908,  p.  298. 


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animal,  large  numbers  of  harmless  bacteria  are  present  in  mouth  and 
intestinal  tract  which  frequently  suppress  further  development  of  single 
invaders  of  a dangerous  kind.  In  addition,  the  healthy  animal  organism 
itself  has,  especially  in  its  blood,  various  means  of  quickly  killing  and 
dissolving  bacteria  which  otherwise  might  cause  a disease. 

At  present  an  antagonistic  process  is  being  extensively  discussed  in 
scientific  as  well  as  in  popular  articles  which  is  characterized  by  a speedy 
dissolution  of  certain  bacteria,  caused  by  an  agent  or  agents  not  yet  defi- 
nitely known.1  A French  investigator,  d’llerelle,  ascribes  this  effect  to  an 
“invisible”  living  organism,  which  he  calls  bacteriophage  (that  isbacteria- 
eater),  while  other  authors  are  of  the  opinion  that  soluble  enzymatic  sub- 
stances are  to  be  held  responsible.  Undoubtedly,  various  causes  may  lead 
to  more  or  less  complete  bacteriolysis.  Within  the  animal  body  the  pro- 
tective substances  secreted  by  the  body  itself,  as  well  as  by  its  normal  bac- 
terial inhabitants,  may  exert  their  antagonistic  action.  But  in  addition, 
and  especially  in  water  and  in  soil,  where  similar  baeteriophagous  proc- 
esses occur,2  other  organisms  may  be  active  which  are  of  such  minute 
size  that  they  pass  through  filters  which  retain  bacteria,  and  are  therefore 
nearly  or  entirely  invisible  under  the  microscope.  It  is  to  be  expected, 
however,  that  more  thorough  researches  will  reveal  that  these  “invisible” 
stages  of  growth  are  connected  with  others  clearly  visible  at  2000-fold 
magnification.  Some  observations  made  point  in  this  direction,  and 
they  indicate  furthermore  that  this  bacteriolysis  may  be  either  true 
autolysis  (caused  by  enzymatic  or  other  substances  produced  by  the  bac- 
teria themselves)  or  a genuine  disease  of  the  bacteria  (caused  by  foreign 
organisms).  Both  possibilities  are  supported  by  observations,3  which 
however  need  further  elucidation,  as  does  the  whole  problem  of  bae- 
teriophagy. 

Better  known  and  of  greater  practical  importance  is  the  bacterio- 
phagous  action  of  protozoa  in  the  intestines,  in  water,  and  in  the  soil. 
The  so-called  self-purification  of  water,  as  well  as  certain  types  of  “soil 
sickness,”  are  to  a large  extent  the  result  of  the  elimination  of  great  num- 
bers of  bacteria  by  protozoa,  welcome  in  the  first  case,  but  disadvan- 
tageous in  the  latter. 

1 Most  of  these  papers  were  published  in  Compt.  rend.  Soc.  Biol,  (tomes  83-85) . 
D’Herelle’s  contributions  were  collected  in  a monograph  of  the  Pasteur  Institute  of 
Paris,  entitled  “Le  Bacteriophage”  (1921),  and  a summary  has  been  given  by 
Davison  in  Abstr.  Bad.,  vol.  6.  1922,  p.  159. 

2 J.  Dumas,  Compt.  rend.  Soc.  Biol.,  tome  83,  1920,  p.  1314. 

3 E.  Almquist,  Centralbl.  f.  Bakt.,  I.  Abt.,  Orig.  Bd.  60,  p.  167;  Saltmbent,  Compt, 
rend.  Soc.  Biol.,  tome  83,  1920,  p.  1545;  O.  Bail,  Wiener  klin.  Wochenschr.,  Bd.  34. 
1921,  p.  237;  Ph.  Kuhn,  Berliner  klin.  Wochenschr.,  Bd.  58,  1921,  p.  296;  Pico,  Compt. 
rend.  Soc.  Biol,  tome  87,  1922,  p.  836. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  57 


5tr.  lacfis  + B.  fluoresccns 


Fig.  17. — Multiplication  of  B.  coli,  B.  fluorescens  and  Streptococcus  lactis,  singly  and 
combined,  in  milk  at  3 to  20°  C.  (Numbers  of  bacteria  are  in  thousands  per  cc.) 


58 


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Experiments  on  Symbiosis  and  Antagonism. — The  curves  shown  in 
Fig.  17  illustrate  how  such  actions  may  be  measured  accurately.1  Three 
of  the  most  common  milk  bacteria  ( B . coh,  B.  fluorescens,  and  Strepto- 
coccus lactis)  were  grown  in  milk  at  various  temperatures  separately  and 
combined.  The  multiplication,  observed  at  intervals,  indicates  clearly 
that  B.  coli  (frequent  in  the  intestines  and  in  feces)  is  strongly  sup- 
pressed by  the  common  lactic  acid  bacteria  ( Streptococcus  lactis ) espe- 
cially at  temperatures  above  10°  C.,  while  on  the  other  hand  B.  fluorescens, 
which  digests  casein  and  produces  a slightly  alkaline  reaction,  stimulates 
the  growth  of  Streptococcus  lactis,  and  is  in  turn  itself  stimulated  by  this 
symbiosis. 

Chemical  tests  will  also  often  prove  helpful  to  ascertain  whether  sym- 
biotic or  antagonistic  processes  are  to  be  taken  into  account.  Considering 
the  very  large  number  of  different  species  present  and  jointly  active 
nearly  everywhere,  it  is  indeed  self-evident  that  such  investigations  are 
quite  indispensable  if  correct  ideas  of  these  highly  complicated  functions 
are  to  be  secured. 


4.  RESISTANCE  OF  RESTING  FORMS 

The  resting  forms  of  the  bacteria  are  microcysts,  arthrospores,  and 
endospores ; those  of  the  lower  fungi,  spores  and  c-onidia ; and  those  of 
the  protozoa,  cysts  (see  Chapter  II).  They  are  formed  in  greatest 
numbers  as  soon  as  the  environmental  conditions  begin  to  become  less 
suitable  for  vegetative  growth;  decrease  in  the  food  supply  and  in  the 
water  content  of  the  substrates  exerts  the  most  pronounced  influence 
upon  this  process.  If  no  lack  of  food  and  water  occurs,  as  is  often  the 
case  when  microorganisms  are  grown  in  the  laboratory  (especially  in 
milk),  the  inclination  to  produce  resting  forms  ceases  gradually  and  may 
he  permanently  lost.  After  spores  and  cysts  are  formed  they  are  able 
to  survive  long  periods  in  a dormant  state ; but  as  soon  as  food  supply, 
reaction,  moisture,  and  temperature  are  again  suitable  for  new  vegeta- 
tive growth,  germination  will  take  place. 

Resistance  of  Endospores. — Bacterial  endospores  are  endowed  with 
the  greatest  resistance  against  all  kinds  of  unfavorable  influences.  They 
will  hardly  ever  die  even  under  the  worst  conditions  they  may  encounter 
in  nature.  It  needs  man’s  action  to  kill  them.  Complete  dryness  and 
lowest  temperatures  are  without  any  effect.  Many  seeds  of  higher  plants 
show  a similar  behavior.2 

1 The  countings  used  for  constructing  the  curves  were  made  by  W.  B.  Lfxwolda. 
Centralbl.  f.  Bakt.,  II.  Abt.,  Bd.  31,  1911,  pp.  129-174. 

2P.  Becqtjerel,  Compt.  rend,  Acad,  Paris,  tome  148,  1909,  p.  1052;  tome  150, 
1910,  p.  1437. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  59 


Live  steam  (of  nearly  100°  C.)  kills  bacterial  spores  usually  after 
1 to  10  minutes;  some  die  even  when  temperatures  of  90°  to  95°  C.  are 
reached.  Others,  however,  are  much  more  resistant,  especially  those  of  the 
so-called  hay  and  potato  bacilli,  normally  present  in  large  numbers  upon 
hay,  straw,  and  in  soil.  They  are  able  to  withstand  the  action  of  flowing 
steam  for  15  to  20  hours  and  more,  but  are  soon  killed  if  the  temperature 
of  the  steam  is  raised  to  125°  to  135°  C.  and  its  pressure  to  about  20  to  30 
lbs.  (IV2  to  2 atmospheres) . Dry  air  must  be  heated  up  to  170°  to  180°  C. 
before  the  same  result  is  attained.  Alfalfa  seeds  were  also  found  to  be 
able  to  survive  moist  heat  of  100°  C.  for  several  hours,  of  120°  C.  for 
31/2  hours. 

If  the  bacterial  spores  are  covered  by  protective  substances  their  re- 
sistance may  go  much  higher.  That  is  especially  the  case  when  they  are 
distributed  in  soil,  wherein  they  can  be  killed  only  by  very  intense 
and  prolonged  heating.  Occasionally  truly  astonishing  results  were 
obtained  with  certain  strains  of  the  potato  bacillus  ( Bac . mesentericus ) 
immediately  after  isolation  from  the  waste  lime  of  sugar  factories.  The 
spores  survived  in  this  case  hot  air  of  310°  to  320°  C.  for  30  minutes, 
live  steam  for  25  hours,  boiling  in  4 per  cent  caustic  soda  or  in  4 per  cent 
hydrochloric  acid  for  20  to  30  minutes.1 

Resistance  of  Other  Resting’  Forms. — Mold  spores  and  conidia  are 
generally  less  resistant  than  bacteria  spores  against  high  temperatures, 
but  sometimes  they  come  fairly  close  to  them.  Arthrospores  and  micro- 
cysts of  bacteria  are  usually  killed  if  the  solution  wherein  they  are  sus- 
pended is  heated  up  to  75  to  95°  C.  Least  resistant  are  most  of  the 
cysts  of  protozoa  for  which  the  upper  limit  is  at  60  to  70°  C.,  while  the 
thermal  death  point  for  the  non-encysted  protozoa  is  in  the  neighborhood 
of  only  50°  C.2 

But  high  temperatures  do  not,  as  a rule,  endanger  bacterial  life 
under  natural  circumstances.  Lack  of  food  and  water,  as  well  as 
low  temperatures,  are  practically  the  only  harmful  physical  factors  in 
nature,  harmful,  however,  only  for  vegetative  cells.  All  resting  forms 
are  sufficiently  protected  to  resist  their  influences  and  to  safeguard  the 
continuity  of  bacterial  life  under  all  circumstances. 

The  regenerative  bodies  of  bacteria,  although  not  true  resting  forms, 
may  also  participate  in  the  conservation  and  continuation  of  bacterial 
life.  Dry  periods  and  frost  do  not  harm  them  in  any  degree,  and  even 
against  relatively  high  temperatures  a rather  strong  resistance  is  shown 
occasionally.  In  heating  tests  this  has  repeatedly  led  to  quite  unex- 

1 Zettnow,  Centralbl.  f . Bakt.,  I.  Abt.  Orig.,  Bd.  66,  1912,  p.  131. 

2 A.  Cunningham  and  F.  Lohnis,  “Studies  on  Soil  Protozoa,”  Centralbl.  f.  Bakt., 
II.  Abt.,  Bd.  39,  1913,  p.  596. 


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pected  results,  because  thus  far  the  presence  of  such  bodies  has  usually 
been  overlooked.1 

6.  DISTRIBUTION  OF  MICROORGANISMS  IN  NATURE 

The  exceptional  ability  of  bacteria  and  other  microorganisms  to  make 
use  of  and  to  adapt  themselves  to  widely  differing  and  varying  environ- 
mental conditions  explains  the  fact  that  they  are  of  a truly  cosmopolitan 
character.  They  are  more  generally  present  on  our  planet  than  are 
higher  plants  and  animals.  Because  of  their  minute  size  they  are  car- 
ried by  wind  and  dust  practically  everywhere,  and  wherever  residues  of 
higher  life  are  to  be  decomposed  these  highly  efficient  destructive  agents 
are  present,  ready  to  do  their  work.  According  to  circumstances  the 
microflora  and  the  microfauna,  that  is  the  entirety  of  microscopic  plants 
and  animals  growing  at  a certain  location,  differ  in  quality  as  well  as  in 
quantity,  and  with  changing  conditions  more  or  less  far-reaching  altera- 
tions take  place  in  order  to  re-establish  an  equilibrium  between  micro- 
flora and  microfauna  and  their  environment. 

Germ  Content  of  Soil. — On  and  in  the  soil  originates  primarily  the 
life  of  all  higher  as  well  as  of  all  lower  organisms.  Fertile  surface  soils 
are  especially  rich  in  rod-like,  motile,  and  sporulating  bacteria.  Psy 
chrophilie  species  predominate  in  cold  climates,  thermophilic  in  the  sub- 
tropics and  tropics.  In  soils  of  approximately  neutral  reaction  bacteria 
are  more  numerous  than  fungi ; in  acid  humus  soils  the  contrary  holds 
true.  Protozoa  and  lower  algae  are  also  to  be  found  in  nearly  every  soil ; 
but  only  where  the  water  supply  is  comparatively  high  and  regular  (as  in 
greenhouses  and  in  irrigated  fields)  are  these  two  groups  of  organ- 
isms more  or  less  abundant.  In  average  field  soils  about  50,000  to 
100,000  of  the  latter  kinds  are  usually  present  in  1 g.  soil,  besides  one  to 
several  hundred  thousands  of  molds,  and  a few  to  many  millions  of  bac- 
teria. Not  infrequently  100  million  bacteria  are  found  in  1 g.  soil,  where 
they  nearly  always  exert  the  greatest  activity.  The  other  microorganisms 
taken  together  may  represent  a larger  volume  of  living  matter,  but 
usually  a less  active  total  surface,  because  of  the  differences  in  size  dis- 
cussed in  Chapter  I (p.  17). 

One  hundred  million  of  bacteria  in  1 g.  of  soil  seems  to  be  a surpris- 
ingly great  number ; in  fact,  however,  they  are  not  very  many  compared 
with  the  space  they  occupy.  One  hundred  millions  in  1 g.  of  soil  are  equal 
to  100,000  in  1 mg.  One  gram  of  soil  fills  approximately  1 cc.,  and  1 mg. 
1 cubic  millimeter.  One  thousand  million  bacteria  fill  the  latter  space,  if 

1 Lohnis,  ‘'Studies  upon  the  Life  Cycles  of  the  Bacteria:  Part  I,”  Memoirs  of  the 
National  Academy  of  Sciences,  vol.  XVI,  No.  2,  pp.  131,  136,  and  143. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  61 


lying  closely  together.  Accordingly,  the  100,000  bacteria  in  1 cubic  milli- 
meter of  soil  occupy  only  1/10,000  of  the  total  space  available.  If  these 
bacteria  were  evenly  distributed  in  the  soil,  and  the  latter  could  be  ex- 
amined in  situ  at  1000-fold  magnification,  a picture  would  become  visible 
similar  to  that  shown  in  Fig.  18.  In  reality,  however,  the  bacteria  are 
mostly  accumulated  in  colonies  within  the  soil,  and  the  sterile  stretches 
lying  between  these  settlements  are  therefore  still  much  wider. 

But  if  the  weight  of  these  living  organisms  is  taken  into  account,  quite 
impressive  figures  result.  One  hundred  million  bacteria  weigh  approxi- 
mately 1/10  mg.,  and  1 acre  of  surface  soil  of  30  cm.  (12  in.)  depth  about 
4 million  lbs.  One  acre  contains,  therefore,  about  350  lbs.  of  living  bac- 
teria, besides  175  to  350  lbs.  of  fungi,  protozoa,  and  algae,  altogether  525 
to  700  lbs.  per  acre.  The  weight  of  cattle  kept  on  a pasture  is  nearly 
equal  to  the  weight  of  microorganisms  in  the  soil  beneath. 


Fig.  18. — Schematic  sketch  of  soil  1000-fold  magnified,  containing  100  million  bacteria 
per  g.  Arrows  indicate  the  location  of  the  bacteria. 


Generally  most  of  the  bacteria  are  to  be  found  at  a depth  of  about 
10  to  15  cm.  below  the  surface.  In  wet  soils  the  upper  layers  are  pre- 
ferred ; in  arid  soils  deeper  strata  are  also  rich  in  bacterial  life.1  But 
lack  of  food  and  air  tends  to  reduce  and  to  exclude  germ  life  in  greater 
depths,  although  in  old  geological  deposits,  of  course,  traces  may  be  found 
which  indicate  that  myriads  of  years  ago  bacteria  have  been  as  active  as 
they  are  to-day  in  disintegrating  all  residues  left  by  higher  organisms. 
Renault  and  other  French  authors2  have  made  special  studies  upon  this 
problem,  which,  however,  will  never  be  definitely  solved. 

1 C.  B.  Lifman,  “The  Distribution  and  Activities  of  Bacteria  in  Soils  of  the  Arid 
Region,”  Univ.  Calif.  Pubs,  in  Agric.  Sciences,  vol.  1,  1912,  pp.  1-20. 

2 Renault’s  numerous  contributions  appeared  from  1894-1900  in  the  Compt.  rend. 
Acad.  Paris,  Annales  des  sciences  naturelles,  and  other  publications.  See  also  Baudouin, 
Compt.  rend.  Acad.  Paris,  vol.  138,  1904,  p.  1001. 


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Germ  Content  of  Water. — According  to  the  amount  of  organic  mat- 
ter present  in  water  very  numerous  or  only  very  few  microorganisms  may 
be  found.  Pure  water  of  deep  wells  is  nearly  sterile,  but  water  of  muddy 
rivers  is  naturally  rich  in  all  kinds  of  microorganisms.  In  the  ocean 
more  bacteria  live  close  to  the  shore  than  on  the  high  sea  or  at  great 
depths.  For  drinking  water  100  bacteria  per  c-.c.  was  often  considered  a 
maximal  number  which  should  not  be  surpassed.  But  it  goes  without 
saying  that  in  this  as  in  all  cases  the  quality  of  the  microorganisms,  not 
their  quantity,  is  the  decisive  factor.  One  cholera  or  typhoid  germ,  of 
course,  makes  water  highly  dangerous  no  matter  how  low  its  total  germ 
content  may  be.  Mineral  waters,  lemonades,  and  other  so-called  soft 
drinks  contain  quite  frequently  large  numbers  of  bacteria,  even  if 
they  are  impregnated  with  carbon  dioxide.1  In  ponds  and  rivers  numer- 
ous algae  and  protozoa,  besides  bacteria,  are  to  be  found;  they  are  true 
water  organisms  and  contribute  to  the  so-called  self-purification  of  such 
waters. 

Germ  Content  of  Air. — Drainage  waters  carry  bacteria  from  the  soil 
into  wells  and  rivers;  wind  and  dust  lift  them  from  the  ground  into  the 
air.  As  no  such  possibility  exists  on  the  high  sea,  on  glaciers,  and  on 
arctic  snow  fields,  the  air  of  those  regions  is  practically  free  of  germs. 
Rain,  snow,  and  hail  stones  contain  variable  numbers  of  microorganisms, 
dependent  on  the  amount  of  dust  in  the  air,  the  length  of  time  during 
which  no  rain  was  falling,  and  on  the  bactericidal  effect  of  bright  sun- 
shine.2 Accordingly,  the  germ  content  of  air  in  large  cities  is  generally 
higher  than  in  rural  districts,3  although  heavy  automobile  traffic  over  dirt 
roads  may  change  this  relation  entirely.  Usually  the  air  in  stables  is 
highly  polluted,  especially  if  dusty  hay  and  straw  are  used,  and  no 
adequate  provision  is  made  for  light  and  ventilation.  This  fact  is  of 
considerable  importance  in  the  production  of  clean  milk.  Globular 
cells  (micrococci,  mold  spores,  and  cysts  of  protozoa)  are  generally  more 
numerous  in  the  air  than  are  rod-shaped  cells,  because  the  latter  are  less 
easily  kept  afloat  by  slight  drafts  of  air. 

Germ  Content  of  Plants. — That  many  soil  organisms  are  to  he  found 
on  growing  plants  needs  no  explanation.  But  in  addition  to  this  acci- 
dental microflora  there  is  another  more  specific  one.  Young  plants  raised 
in  a germ-free  environment  show  this  clearly;  the  particular  bacteria  at- 

1 IIochstetter,  Arb.  a.  d.  kais.  Gesundh.  Amte,  Bd.  2,  1SS7,  p.  1;  Thoxi,  Cen- 
tralhl.f.  Bakt.,  II.  Abt.,  Bd.  29,  1911,  p.  616. 

2 Flemming,  Zeitschr.f.  Hyg.,  Bd.  58,  1908,  p.  345. 

3P.  Miquel,  Annuaire  de  VObservatoire  de  Monlsouris,  1882;  Saito,  Jour.  CoU. 
Science,  Imper.  Univ.,  Tokyo,  vol.  23,  1908. 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  63 


tached  to  the  seed  multiply  rapidly  and  cover  the  whole  plant  with  an  al- 
most continuous  thin  slimy  layer  of  bacteria.  The  slime  produced  by 
them  not  only  prevents  their  being  -washed  off  by  heavy  rains,  but  also 
helps  to  preserve  a sufficient  amount  of  moisture  even  during  periods  of 
drought.  Besides  dew,  small  amounts  of  sap  excreted  by  the  plants  are 
available  to  the  bacteria.  Such  sap  contains  0.05  to  0.1  per  cent  organic 
and  inorganic  substances,  quite  enough  for  these  modest  organisms.  Dry- 
ing (of  hay,  straw,  etc.)  kills  only  part  of  them,  while  storage  of  fresh 
material,  especially  if  fermentation  takes  place  as  in  the  silo,  is  usually 
accompanied  by  a considerable  increase  in  number,  1000  to  2000  millions 
per  g.  being  no  rare  occurrence  in  such  cases.  Because  composition  and 
reaction  of  the  sap  varies  with  the  different  plants,  their  specific  micro- 
flora  varies  accordingly.  Corn  and  cabbage,  for  instance,  are  rich 
in  lactic  acid  bacteria,  and  therefore  especially  suited  to  undergo  an 
acid  fermentation  in  the  silo  and  in  the  sauerkraut  vat.  The  roots,  too, 
have  their  special  microflora,  and  this  may  contribute  to  the  favorable 
or  unfavorable  effects  noticeable  with  certain  crops  in  successive 
plantings. 

Microorganisms  on  and  in  Animals. — A young  animal  before  birth 
is  practically  sterile,  and  can  be  kept  so  if  taken  from  the  mother  by 
hysterotomy.  In  the  normal  course,  however,  a varied  microflora  soon  es- 
tablishes itself  on  the  skin  and  within  the  intestinal  tract.  Species  of 
bacteria  adapted  to  higher  temperature  and  to  low  oxygen  tension  find 
such  conditions  most  favorable.  In  the  first  stomach  of  ruminants  rapid 
multiplication  takes  place,  sometimes  accompanied  by  liberation  of  large 
amounts  of  gases ; but  later,  after  the  acid  gastric  juice  has  been  added  in 
the  fourth  division,  a more  or  less  marked  reduction  in  numbers  becomes 
noticeable.  When  empty,  this  part  of  the  digestive  tract,  as  well  as  the 
small  intestines,  is  practically  sterile;  in  the  latter  case  the  effect  of  the 
acid  is  strengthened  by  a direct  bactericidal  action  of  the  mucous  lining. 
But  a profound  change  occurs,  and  a rapid  multiplication  of  bacteria  and 
protozoa  starts  again  in  the  last  part  of  the  intestinal  tract.  Again  gases 
and  offensive  odors  give  testimony  of  presence  and  activity  of  numerous 
microorganisms.  Not  less  than  10  to  20  per  cent  of  the  dry  matter  in 
feces  is  made  up  of  living  and  dead  bacteria.  "Up  to  18,000  millions  per  g. 
have  been  found  alive  in  solid  excreta ; fresh  urine,  on  the  other  hand,  is 
nearly  sterile,  but  soon  becomes  strongly  con+aminated  on  the  ground. 

On  the  skin  numerous  microorganisms  are  continually  deposited  from 
the  air,  the  litter,  and  the  dung.  They  multiply  in  the  presence  of  suffi- 
cient moisture ; perspiration  is  of  importance  in  this  respect.  Each  gram 
of  dirt  removed  by  grooming  contains  several,  frequently  hundreds  of 


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millions  of  bacteria,  and  it  is  obvious  that  by  touching  the  flanks  or  the 
unclean  udder  of  a cow  many  bacteria  will  be  transported  by  the  hands 
of  the  milker  into  the  milk  pail.  Comparatively  few  microorganisms 
make  their  way  through  lesions  of  the  outer  skin  or  through  the  mucous 
membranes  of  the  digestive  tract  into  the  inner  parts  of  the  body,  which 
in  a state  of  health  are  practically  sterile.  The  bactericidal  action  of  the 
blood,  first  observed  by  Edm.  King  (see  p.  7),  plays  an  important  role 
in  this  respect.  How  and  why  pathogenic  organisms  may  overcome  this 
action  will  be  discussed  in  Chapter  VII,  6. 

Germ  Content  of  Milk  and  Dairy  Products. — Milk  produced  in  a 
healthy  udder  is,  at  first,  free  of  germs.  But  contamination  takes  place 
before  the  milk  leaves  the  udder.  The  teats  with  the  small  milk  droplets 
left  there  from  each  milking  enable  the  bacteria,  thriving  in  soiled  litter, 


Fig.  19. — Schematic  sketch  of  sour  milk  1000-fold  magnified,  containing  1000  million 
bacteria  per  cc.,  distributed  between  the  lighter  stained  flakes  of  casein. 

to  invade  the  udder ; and  though  on  account  of  the  bactericidal  action  of  the 
healthy  body  tissue  most  of  them  do  not  get  a firm  foothold,  certain  kinds 
prove  sufficiently  resistant  to  develop  a specific  microflora  in  the  ducts  of 
the  udder.  Every  time  when  the  milk  is  drawn  from  the  udder  some  of 
these  bacteria  are  washed  out,  acting  as  an  initial  contamination.  But 
they  mean  little  compared  with  the  large  numbers  and  various  kinds 
brought  into  the  milk  with  falling  dirt,  by  contact  with  the  milk  pail  and 
other  utensils,  especially  if  these  are  not  scrupulously  clean,  that  is, 
sterilized.  As  milk  is  rich  in  nutrients,  rapid  multiplication  takes  place 
as  long  as  its  temperature  remains  comparatively  high  (see  p.  26)  ; quick 
cooling  is  therefore  of  very  great  importance.  If  all  possible  precaution- 
ary measures  are  applied,  the  total  germ  content  of  milk  can  be  kept  at  a 
few  hundreds  per  cc.  Ordinary  market  milk  harbors  always  1 to  10 
or  more  millions  per  cc.,  which  may  be  killed  but  not  removed  by  pasteuri- 
zation in  the  dairy  or  by  boiling  in  the  household.  When  milk  turns  acid 
1000  to  2000  millions  of  bacteria  are  usually  present  in  each  cc. ; such 


RELATIONS  OF  MICROORGANISMS  TO  THEIR  ENVIRONMENT  65 


milk,  1000-fold  magnified,  would  present  a picture  like  Fig.  19,  which 
should  be  compared  with  Fig.  18.  Despite  the  apparently  very  large 
number  of  bacteria  present,  most  space  is  still  occupied  by  the  milk 
itself.  Generally  the  same  holds  true  with  regard  to  butter  and  cheese, 
as  was  shown  in  Fig.  6 (p.  18).  Occasionally,  however,  as  in  the  whitish 
slimy  surface  layer  characteristic  of  certain  kinds  of  young  cheese,  a 
fairly  solid  layer  of  bacterial  and  fungous  cells  may  occur,  made  up  of 
approximately  500,000  million  cells  per  g. 

Germ  Content  of  Manure. — Compared  with  the  very  large  number 
of  bacteria  present  in  the  solid  excreta,  those  of  litter  and  urine  are  al- 
most negligible.  But  while  in  the  feces  practically  all  residues  are  al- 
ready brought  to  an  advanced  state  of  decomposition,  there  is  still  much 
material  left  in  litter  and  urine  which  is  of  value  to  these  bacteria.  Fa- 
vored by  the  higher  temperature  in  stables  and  manure  piles,  new  multi- 
plication starts  quickly  and  after  some  days  or  weeks  every  gram  of  the 
mixture  contains  several  thousand  millions  of  bacteria,  fungi,  and  pro- 
tozoa.1 If  the  weight  of  these  organisms  is  compared  with  that  of  the 
manure  spread  on  the  field,  it  follows  that  in  20  tons  of  manure  per 
acre  approximately  450  lbs.  of  living  matter  is  being  added  to  the  soil. 
Only  part  of  these  dung  bacteria  will  continue  to  grow  vigorously  in  the 
new  environment,  while  others  will  die;  but  it  becomes  at  once  evident 
that  this  special  effect  of  an  application  of  animal  manure  must  be  of 
great  importance  on  all  soils  which  are  not  yet  enriched  in  bacterial  life 
by  long  and  careful  cultivation.  Where  hot  and  dry  seasons  tend  to  re- 
duce bacterial  life  in  the  soil,  regular  application  of  well-rotted  stable 
manure  are  especially  to  be  recommended. 

Adaptation  to  the  Environment. — The  cycle  of  matter  which  starts 
and  ends  in  the  soil  is  accompanied  throughout  its  course  by  microorgan- 
isms which  also  originate  in  the  soil  and  return  to  it.  On  their  way  they 
are  exposed  to  widely  varying  environmental  conditions,  which  not  only 
cause  alterations  in  number  and  species,  but  also  lead  to  adaptations  and 
to  the  development  of  new  variations  best  suited  to  grow  in  this  new  sur- 
rounding. Occasionally  such  local  varieties  of  microorganisms  are  of 
great  practical  importance,  especially  in  the  manufacture  of  butter  and 
cheese,  where  they  may  be  used  as  selected  pure  cultures.  But  the  detri- 
mental effects  exerted  by  certain  kinds  of  manuring  and  feeding  upon  the 
quality  of  dairy  products  are  also  in  part  known  to  be  caused  by  such 
special  varieties  of  microorganisms. 

1 F.  Lohnis  und  J.  H.  Smith,  “Die  Veranderungen  des  Stalldungers  wahrend  der 
Lagerung  und  seine  Wirkung  im  Boden,”  Fuhlingslandw.  Zeitg.,  Bd.  63,  1914,  pp.  153- 

167. 


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Because  of  the  close  connections  existing  between  the  bacterial 
growths  in  soil,  on  plants,  in  air,  water,  milk,  dairy  products,  and  in 
manure,  all  fundamental  studies  in  agricultural  bacteriology  must  take 
these  relations  into  account  and  must  be  planned  accordingly.  Only  upon 
such  a basis  is  it  possible  to  investigate  successfully  the  more  specialized 
problems  of  dairy  and  soil  bacteriology. 


CHAPTER  V 


COUNTING,  ISOLATING,  CULTIVATING,  AND  TESTING 
BACTERIA  AND  RELATED  MICROORGANISMS 

In  order  to  get  accurate  estimates  of  number  and  kinds  of  microor- 
ganisms present  and  active  in  soil,  water,  milk,  manure,  etc.,  various 
methods  of  counting,  isolating,  and  testing  bacteria,  fungi,  and  protozoa 
have  been  developed.  Most  of  them  are  not  very  complicated,  and  it  is 
possible  within  a comparatively  short  time  to  acquire  enough  technical 
skill  to  make  such  investigations.  A certain  amount  of  laboratory  work 
is  indeed  quite  indispensable  for  acquiring  correct  ideas  in  regard  to 
bacteriological  problems.  But  the  relative  simplicity  of  bacteriological 
technique  should  certainly  not  create  the  erroneous  impression  that  a 
few  weeks’ or  months’ training  would  be  sufficient  preparation  for  solving 
the  most  difficult  problems.  In  addition  to  technical  skill,  a thorough 
knowledge  of  the  bacteriological  and  agricultural  literature,  clear,  criti- 
cal thinking,  exact  observation,  and  much  persistence  are  needed  to  at- 
tain really  valuable  results.  Merely  the  rough  outlines  of  bacteriological 
technique  are  given  on  the  following  pages.  More  detailed  information 
may  be  gathered  from  the  books  mentioned  on  p.  11  under  C and  D. 

Counting  Bacteria  and  Fungi. — The  very  large  number  of  microor- 
ganisms present  in  soil,  manure,  milk,  etc.,  always  makes  it  necessary  to 
work  with  comparatively  small  amounts  of  material.  But  because  in  most 
cases  the  organisms  are  rather  irregularly  distributed  and  congregated 
in  different  parts  of  the  substrate,  large  samples  are  to  be  taken,  at  first, 
from  which  after  careful  mixing,  gradually  smaller  and  smaller  samples 
are  prepared,  if  necessary,  by  diluting  the  substances  with  water  or  other 
liquids,  wherein  all  living  cells  have  been  previously  killed  by  boiling. 
With  the  smallest  samples  the  counting  is  made  either  directly  under  the 
microscope,  or  indirectly  by  growing  the  microorganisms  in  solid  or  in 
liquid  substrates.  Each  of  these  methods  has  its  advantages  and  disad- 
vantages. 

Microscopic  counts  have  become  very  useful  for  determining  the  germ 
content  of  milk.  One  one-hundredth  cubic  centimeter  is  evenly  spread  on 
a measured  space  of  a glass  slide,  usually  1 cm.2,  dried,  stained,  and 
examined  at  1000-fold  magnification.  The  cells  visible  in  every  one  of 

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twenty  or  more  fields  are  counted,  and  the  average  number  of  these 
counts  is  multiplied  with  a factor  which  correlates  the  size  of  the  micro- 
scopic field  with  the  area  covered  by  1/100  cc.  of  milk  and  with  1 cc., 
respectively.  If  sufficient  parallel  tests  are  made,  and  the  germ  content 
of  the  milk  is  not  too  low,  the  figures  obtained  are  fairly  accurate ; fur- 
thermore, not  merely  a bare  number  is  secured,  but  also  a certain  amount 
of  information  concerning  the  quality  of  the  microflora  in  that  particular 
milk.  The  main  disadvantage  is  that  besides  living  bacteria  a smaller  or 
larger  number  of  dead  organisms  may  be  visible;  sometimes  they  are 
more  weakly  stained  and  can  so  be  differentiated,  but  this  is  not  always 
true.  Such  heterogeneous  and  insoluble  materials  as  manure  and  soil 
are  of  course  not  suited  for  direct  microscopic  tests.  It  is  impossible  to 
get  an  unobstructed  view  of  the  bacteria;  many  of  them  are  hidden  be- 
hind and  within  the  irregular  particles  of  these  substances. 

Cultural  tests  have  the  advantage  that  they  are  applicable  in  all 
cases,  but  the  disadvantage  that  not  all  organisms  which  are  present  will 
grow  on  the  substrates  used.  In  most  cases  solid  substrates  find  appli- 
cation, and  the  number  of  colonies  developing  on  them  is  used  as  basis 
for  calculating  the  number  of  viable  bacteria  and  fungi  present  in  the 
material  tested.  The  Petri  dish  shown  in  Plate  III  contained,  for  in- 
stance, the  colonies  which  grew  from  1/1,000,000  g.  soil.  However,  the 
numbers  obtained  in  this  way  are  always  below  the  actual  number  of 
viable  germs  present.  Their  requirements  are  too  different  to  permit 
growth  of  all  of  them  on  the  same  substrate  at  the  same  temperature  in 
the  presence  or  absence  of  air.  To  make  them  all  grow,  many  substrates 
of  various  composition  and  of  different  reactions,  cultivation  at  high  and 
low  temperatures,  under  aerobic  and  anaerobic  conditions  would  have 
to  be  used.  Furthermore,  many  colonies  are  not  the  offspring  of  a single 
cell,  but  of  a small  or  large  clump  of  organisms.  Certain  groups  of  or- 
ganisms do  not  grow  at  all  on  the  solid  substrates  commonly  used.  In 
such  cases  liquid  media  of  suitable  composition  are  inoculated  with  a 
series  of  dilutions  made  from  the  original  material,  and  it  is  then  deter- 
mined at  what  dilution  the  development  ends.  Heating  of  the  substrates 
to  80  to  90°  C.  immediately  after  inoculation  permits  counting  of  bac- 
terial spores  and  other  resting  forms.  For  enumerating  fungus  germs 
solid  substrates  of  acid  reaction  are  most  convenient. 

Counting  Protozoa. — Microscopic  as  well  as  cultural  tests  are 
equally  applicable  to  protozoa.  Because  most  of  them  are  larger  than 
the  bacteria,  microscopic  counts  give  fairly  satisfactory  results  even  with 
soil  and  manure.  But  in  most  cases  the  dilution  method  with  solutions 
of  various  composition  kept  at  different  temperatures  proves  preferable. 
Microscopic  examinations  of  these  solutions  give  a good  survey  of  the 


COUNTING,  CULTIVATING,  AND  TESTING  BACTERIA 


69 


various  groups  of  protozoa  present.  Preliminary  treatment  of  the  ma- 
terial with  hydrochloric  acid  makes  it  possible  to  enumerate  only  the 
encysted  forms  and  to  subtract  their  number  from  the  total  counts.1 

Isolating'  Bacteria,  Fungi,  and  Protozoa. — The  solid  and  liquid  sub- 
strates used  for  counting  microorganisms  permit  also  their  being  isolated 
in  “pure  culture.”  The  first  growth  obtained  from  any  given  material 
is  practically  never  pure,  but  a mixture  of  various  kinds  of  organisms.  If 
solid  substrates  are  used  the  different  appearances  of  the  colonies  demon- 
strate this  fact  clearly.  In  the  case  of  liquid  substrates  microscopic  tests 
furnish  analogous  evidence.  But  it  is  merely  necessary  to  repeat  the 
procedure  with  these  “crude”  cultures  first  obtained  until  ultimately 
only  one  type  of  colonies  and  one  type  of  cells  remain.  At  the  time  of 
Louis  Pasteur  liquid  substrates  alone  were  used,  as  a rule  with  unsatis- 
factory results.  Solid  substrates,  introduced  by  Robert  Koch,  are  un- 
doubtedly superior  in  most  cases.  In  fact,  practically  all  pure  culture 
work  was  done  with  them,  and  this  was  the  basis  of  modern  bacteriology. 
Nevertheless,  liquid  cultures  may  occasionally  prove  helpful,  especially 
in  connection  with  the  direct  isolation  of  single  cells. 

Liquid  Cultures. — Liquids  of  different  composition  are  the  natural 
habitat  of  bacteria  and  protozoa  in  soil,  manure,  milk,  etc.  If  the  various 
food  requirements  are  taken  into  account  an  almost  unlimited  number  of 
differently  composed  substrates  may  be  evolved,  which  are  especially 
suitable  for  the  growth  of  one  or  another  group  of  microorganisms.  If  a 
complex  mixture  of  bacterial  species,  as  in  soil,  is  transferred  into  such 
a solution  a natural  selection  takes  place;  most  species  remain  dormant 
and  die  after  a while,  but  those  adapted  to  the  conditions  offered  show 
vigorous  growth.  Professor  M.  W.  Beijerinck  in  Delft,  Holland,  has 
made  most  ingenious  use  of  this  principle,  and  has  discovered  various  im- 
portant groups  of  organisms  by  means  of  such  “accumulation”  experi- 
ments. Pure  cultures,  however,  are  hardly  ever  obtained  by  this  method, 
because  no  single  species  is  so  highly  specialized  that  it  alone  will  grow 
under  certain  conditions,  and  even  the  weakest  dilutions  usually  contain 
not  only  one,  but  several  organisms.  However,  as  a first  step  in  isolating 
bacteria  and  protozoa,  the  use  of  such  “elective  cultures”  will  always  be 
of  great  value. 

Plate  Cultures. — Gelatin  (prepared  from  bones),  agar  (a  partially 
transparent  jelly  made  from  algae  and  composed  mostly  of  carbohy- 
drates), and  silica  jelly  are  the  substances  most  frequently  used  for  con- 
verting liquid  into  solid  substrates.  Gelatin  is  rich  in  nitrogen  and 
easily  liquefied  by  many  bacteria ; its  addition  changes  the  general  char- 

1 D.  W.  Cutler,  Jour.  Agric.  Science,  vol.  10,  1920,  p.  135. 


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acter  of  the  media  considerably.  Agar  exerts  much  less  influence  in  this 
direction,  and  silica  jelly  practically  none.  According  to  circumstances 
one  or  the  other  material  deserves  preference.  When  gelatinous  sub- 
strates were  first  used  in  bacteriological  laboratories  it  was  customary  to 
spread  them  on  simple  glass  plates  aird  to  call  such  cultures  “plate  cul- 
tures.” Although  it  did  not  take  a long  time  to  find  out  that  glass  dishes 
or  flasks  are  much  more  satisfactory,  especially  because  of  the  better 
protection  afforded  against  contamination  from  the  air,  etc.,  the 
term  plate  culture  has  been  generally  retained.  Because  the  jelly  pre- 
vents locomotion  of  motile  bacteria,  and  the  added  nutrient  solution  per- 
mits rapid  multiplication  of  those  organisms  whose  food  requirements 
are  in  accordance  with  it,  colonies  appear  which  may  be  the  progeny  of 
one  or  of  several  cells.  Repeated  platings  and  parallel  tests  lead  sooner  or 
later  to  really  pure  cultures.  Thus  far  it  has  been  an  almost  universal  belief 
that  in  accordance  with  the  monomorphistic  point  of  view,  as  discussed  in 
Chapter  I,  all  colonies  and  all  cells  had  to  present  a uniform  appearance, 
if  a culture  was  to  be  accepted  as  pure.  During  the  last  few  years,  how- 
ever, an  ever  increasing  number  of  observations  has  accumulated,  proving 
beyond  doubt  that  like  the  cell  form,  so  also  the  appearance  of  the  col- 
onies of  pure  cultures  is  not  as  constant  as  was  assumed.  It  goes  without 
saying  that  this  lack  of  constancy  leads  to  new  uncertainties  and  diffi- 
culties. Parallel  tests  and  careful  repetition  of  the  platings  will  prove 
helpful  in  such  cases,  which  fortunately  are  not  very  frequent.  Usually 
fairly  uniform  colony  growth  is  shown  by  pure  cultures  when  originating 
from  young  cells.  For  thorough  investigations  such  growth  should  al- 
ways be  selected. 

Single-Cell  Cultures. — Theoretically  the  ideal  method  of  getting 
strictly  pure  cultures  is  the  direct  isolation  of  single  cells  and  their 
propagation  in  the  absence  of  any  contamination.  With  comparatively 
large  microorganisms,  such  as  yeasts,  molds,  and  bacteria  of  several  micra 
length,  single-cell  cultures  are  not  too  difficult  to  obtain.  Minute  droplets 
of  appropriately  diluted  liquid  cultures  are  placed  on  sterile  cover- 
glasses,  and  after  careful  microscopic  examination  those  droplets  are 
marked  which  happen  to  contain  only  one  single  cell.  Transfers  are  made 
at  once  or  after  the  cell  has  multiplied  in  the  droplet,  which  process  may 
be  watched  microscopically.  With  bacteria  of  the  usual  minute  size  of 
i/2  to  IT/ojU.  this  procedure  is  not  easily  applicable.  In  this  case  the  visi- 
bility of  the  small  cells  suspended  in  the  droplet  may  be  increased  by 
making  the  dilutions  in  India  ink  1 or  similar  liquids,  and  to  place  these 
droplets  upon  a layer  of  gelatin,  where  they  quickly  dry  down.  Those 


1 R.  Burri,  “ Das  Tuschepunktverfahren,  1909. 


COUNTING,  CULTIVATING,  AND  TESTING  BACTERIA 


71 


containing  only  one  cell  are  marked,  and  either  directly  used  for  growing 
pure  cultures  (Fig.  20),  or  transferred  to  other  substrates.  In  addition 
to  these  relatively  simple  methods  several  others  have  been  invented, 
based  on  the  use  of  special  instruments  (capillary  tubes  or  needles)  which 
permit  the  isolation  of  single  cells  directly  under  the  microscope.1  But 
these  last  named  methods  have  not  proved  to  be  very  satisfactory  for 
general  use.  They  require  considerable  technical  skill ; usually  about 
half  of  the  isolated  cells  refuse  to  grow ; and  the  chances  for  contamina- 
tion are  by  no  means  small.  Therefore,  as  a rule,  plate  cultures  are 
practically  much  superior  to  single-cell  cultures,  and  if  the  results 
obtained  are  fully  verified  by  a sufficient  number  of  repeatedly  made 


Fig.  20. — India  ink  droplets  (X500)  containing  (a)  one  cell  and  ( b ) its  progeny. 


parallel  tests  they  are  equally  conclusive,  as  all  carefully  made  com- 
parative tests  of  both  methods  have  shown.2 

Anaerobic  Cultures. — Numerous  procedures  and  kinds  of  apparatus 
have  been  devised  for  isolating  and  cultivating  anaerobic  bacteria.  The 
simplest  and  most  practicable  way  is  undoubtedly  to  remove  the  oxygen  in 
the  cultural  tubes  themselves  by  inserting  a second  cotton  plug  which  is 
moistened  by  pyrogallic  acid,  sodium  hydrosulfite,  or  some  other  oxygen 

1 S.  L.  Schouten,  Zeitschr.  f.  Wissenschcift.  M ikroskopie,  vol.  22,  1905,  p.  10;  vol.  24, 
1907,  p.  258;  M.  A.  Barber,  Kansas  Univ.,  Science  Bulletin,  Vol.  4,  No.  1, 1907;  Jour. 
Infect.  Diseases,  vol.  5,  1908,  p.  379;  vol.  8,  1911,  p.  348;  Jour.  Exp.  Med.,  vol.  32,  1920, 
p.  295.  Hecker,  Jour.  Infect.  Diseases,  vol.  19, 1916,  p.  305;  Hort,  Jour.  Hyg  , vol.  18. 
1920,  p.  361. 

2 Lohnis,  Memoirs  National  Acad.  Sciences,  vol.  XVI,  No.  2,  1921,  p.  39. 


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absorbing  liquid  (Fig.  21b).  The  first  isolation  can  be  conveniently 
made  in  glass  tubes,  as  shown  in  Fig.  21a.  Three  dilutions  made  in  agar 
are  poured  above  each  other,  and  after  the  colonies  have  sufficiently  de- 
veloped, the  rubber  stopper  at  the  lower  end  is  removed,  the  agar  slips 


Fig.  21 —Anaerobic  cultures  (-f  nat.  size),  (a)  Isolating  tubes.  ( b ) Stab  culture. 


out,  and  some  well  isolated  colonies  are  transferred  for  further  examina- 
tion. Vigorous  formation  of  gas  makes  it  sometimes  difficult  to  secure 
well  defined  colonies,  as  the  whole  column  of  agar  is  split  and  torn  in 
such  cases  (Fig.  21a). 


COUNTING,  CULTIVATING,  AND  TESTING  BACTERIA 


73 


Substrates  and  Utensils  for  Bacteriological  Work. — For  accumulat- 
ing as  well  as  for  cultivating  the  various  microorganisms  present  in  soil, 
milk,  manure,  etc.,  numerous  substrates  of  different  composition  must  be 
used.  Plant  decoctions,  whey,  or  extracts  made  from  manure  and  soil 
give  frequently  better  results  than  any  artificial  substrates.  For  com- 
parative tests,  however,  the  latter  are  of  great  importance,  and  no  species 
can  be  accepted  as  properly  described  which  has  not  been  thoroughly 
tested  at  least  on  the  following  media : Beef  agar  without  and  with 


Fig.  22. — Arnold  Sterilizer 
(-^5  nat.  size). 


Fig.  23. — Autoclave  (-^  nat.  size). 


0.5  per  cent  glucose,  beef  gelatine,  beef  broth,  milk,  and  potato.  The  beef 
substrates  are  all  made  from  broth,  prepared  either  from  fresh  meat,  from 
meat  extract,  or  from  dried  media  furnished  by  several  firms.  Details 
to  be  observed  in  the  preparation  of  these  and  of  other  media  are  to  be 
found  in  the  laboratory  manuals  mentioned  under  D on  p.  11. 

After  being  prepared  the  substrates  are  filled  into  test  tubes,  which 
are  closed  with  cotton  stoppers  destined  to  exclude  all  outside  gei’ms.  All 
bacteria  and  molds  present  within  the  media  are  killed  by  thorough  heat- 
ing. This  is  done  either  in  live  steam  in  a sterilizer  of  cylindrical  or 


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cubical  shape  (Fig.  22),  or  under  pressure  in  a so-called  autoclave 
(Fig.  23).  Heating  in  the  autoclave  permits  a more  rapid  and  thorough 
sterilization,  but  not  all  media  can  stand  this  most  severe  treatment. 
For  these,  live  steam  is  to  be  used ; but  on  account  of  the  great  resistance 
of  some  of  the  bacterial  spores,  repeated  heatings,  the  so-called  fractional 
sterilization,  is  required  in  this  case.  After  each  heating  all  or  part 
of  the  surviving  spores  will  germinate,  and  their  progeny  will  then  be 
killed  by  the  next  heating,  usually  on  the  following  day.  If  the  steaming 
is  repeated  at  five  successive  days,  as  a rule  the  media  will  be  sterile, 
although  substrates  exceptionally  rich  in  highly  resistant  spores  may 
still  contain  contaminating  organisms. 

Empty  glass  vessels,  Petri  dishes,  etc.,  are  placed  in  a hot  air 


sterilizer  (Fig.  24)  and  exposed  to  temperatures  of  about  165°  C.  Trans- 
fers of  bacterial  growth  are  made  with  platinum  needles  and  loops,  which 
immediately  before  and  after  use  are  made  red  hot  in  an  open  flame. 

Testing  Pure  Cultures. — Because  of  the  omnipresence  of  microor- 
ganisms it  is  of  preeminent  importance  carefully  to  protect  pure  cultures 
once  obtained  against  all  contaminations.  Sterilized  containers,  sterilized 
media,  and  sterilized  utensils  are  absolutely  necessary.  Parallel  tests  are 
always  to  be  recommended.  The  development  of  pure  cultures  upon  the 
various  substrates  mentioned  above  is  usually  very  characteristic. 
Plate  V.  illustrates  this  fact  with  regard  to  B.  coli,  an  intestinal 
species  and  therefore  common  in  manure  and  in  unclean  milk,  and  B. 
•prodigiosum,  characterized  by  its  red  pigment  best  noticeable  on  agar 
and  on  potato.  Gas  formation  by  B.  coli  is  visible  in  the  glucose  agar 


COUNTING,  CULTIVATING,  AND  TESTING  BACTERIA 


75 


stab  as  well  as  in  milk ; but  only  a trace  of  red  is  produced  on  top  of  the 
milk  culture  of  Bact.  prodigiosum. 

Frequently  such  cultures  are  kept  for  only  a week,  and  many  species 
descriptions  have  been  based  upon  such  short  termed  and  totally  insuffi- 
cient observations.  All  cultures  should  be  tested  at  frequent  intervals 
during  at  least  one  month  and  preferably  longer.  Repeated  tests  are 
necessary  in  order  to  collect  information  upon  constancy  or  variability  of 
the  strains  studied.  Professor  J.  G.  Adami1  once  urged  that  everybody 
who  publishes  a description  of  a new  bacterial  species,  should  furnish  one 
year  later  a second  description  based  on  renewed  studies.  Perhaps  it 
would  be  still  better  to  extend  all  such  investigations  for  a whole  year  or 
longer;  in  this  way  the  careless  “species”  making  would  be  materially 
reduced. 

Microscopic  Studies. — Cultural  tests  should  always  be  accompanied 
by  microscopical  studies,  which  are  not  to  be  restricted  to  one  or  a few 


days,  as  is  frequently  done,  but  they  too  are  to  be  continued  as  long  as 
changes  become  visible  in  order  to  get  a complete  knowledge  of  the 
morphological  characters.  These  tests  are  to  be  made  both  with  living 
unstained  material  suspended  in  water  or  nutrient  solution,  and  with 
dried  and  stained  preparations ; the  latter  are  as  a rule  more  satisfactory, 
especially  with  bacteria.  Plate  I shows  the  different  appearance  of  the 
organisms  when  treated  in  these  various  manners. 

For  observations  of  living  bacteria  so-called  hanging  drop  prepara- 
tions are  most  suitable.  A small  droplet  containing  the  bacteria  is  placed 
on  a coverglass,  and  this  is  fastened  upon  a hollow  slide  so  that  the  drop 
comes  in  the  center,  not  touching  the  sides  nor  the  bottom  of  the  depres- 
sion (Fig.  25).  Especially  at  the  edge  of  the  drop  the  cells  are  clearly 
visible,  and  their  motility  or  immotility  can  be  ascertained.  Some  workers 
prefer  to  have  the  liquid  in  a thin  flat  layer ; this  can  be  accomplished 
by  covering  the  drop  with  a second  coverglass  of  smaller  diameter,  or  by 
placing  a small  piece  of  agar  against  it  (so-called  hanging  agar  block 
preparation) . 

1 Publ.  Health  Papers  and  Rep.,  Amer.  Publ.  Health  Assn.,  vol.  20.  1S94,  p.  415. 


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Stained  preparates  are  usually  made  in  the  following  manner.  The 
bacteria  are  spread  evenly  on  a eoverglass  or  upon  an  ordinary  glass  slide, 
and  allowed  to  become  dry.  They  are  then  fixed  by  quickly  heating  in 
the  open  flame,  or  by  applying  special  fixatives  (alcohol,  osmic  acid,  etc.) 
so  that  they  stick  firmly  to  the  glass,  and  stained  by  pouring  upon  the 
area  covered  by  the  bacteria  an  aqueous  solution  of  some  anilin  dye,  such 
as  methylene  blue,  fuchsin,  Victoria  blue,  gentian  violet,  etc.  After  a 
few  seconds  or  minutes  the  staining  solution  is  poured  off,  the  slide  or  the 
eoverglass  is  thoroughly  rinsed  with  water,  and  after  being  dried  it  is  ready 
for  inspection.  For  fastening  the  eoverglass  upon  a slide  Canada  balsam 
is  mostly  used,  which  becomes  quite  hard  after  a few  days.  Such  mounts, 
if  properly  made,  keep  indefinitely.  Very  convenient,  although  not  al- 
ways applicable,  is  the  method  of  mixing  the  bacteria  with  India  ink 
before  spreading  them  upon  the  eoverglass  or  the  glass  slide.  Such 
smears  need  no  fixing  or  other  treatment,  give  very  clear  pictures,  pro- 
vided that  slime  or  other  disturbing  elements  are  absent,  and  show  de- 
tails within  the  cells  which  in  stained  preparates  can  be  brought  out  only 
by  special,  more  complicated  procedures. 

Up  to  about  500-fold  magnification  the  ordinary  dry  lenses  are  quite 
satisfactory.  For  1000-fold  magnification,  however,  which  is  usually 
necessary  in  bacteriological  work,  some  special  appliances  are  generally 
used  which  were  invented  by  Professor  Ernst  Abbe  at  the  Zeiss  works  in 
Jena,  just  at  the  time  when  Robert  Koch  began  his  important  investiga- 
tions. The  first  one  is  a condenser,  which  concentrates  a large  amount  of 
light  upon  a very  small  part  of  the  preparation,  and  the  second  one  is 
a so-called  immersion  lens,  that  is  a special  type  of  objective  which  is  to 
be  immersed  into  a drop  of  cedar  oil,  placed  upon  the  eoverglass  or 
directly  upon  the  dried  smear.  This  combination  tends  to  direct  as 
much  light  as  possible  into  the  microscope  and  into  the  eye  of  the  ob- 
server, and  assures  therefore  very  clear  and  sharp  pictures,  which  other- 
wise could  not  be  obtained. 

Additional  Tests. — Special  tests  concerning  the  behavior  of  pure 
cultures  at  high  and  at  low  temperatures,  toward  various  sources  of  car- 
bon and  nitrogen,  etc.,  will  often  be  added  advantageously  to  the  ordinary 
cultural  and  microscopical  tests.  A descriptive  chart,  worked  out  and  re- 
vised from  time  to  time  by  a Committee  of  the  Society  of  American  Bac- 
teriologists, furnishes  valuable  details  in  this  direction,  but  again  it  must 
be  strongly  emphasized  that  such  experiments  are  of  real  value  only  if 
they  are  extended  over  long  periods,  checked  by  parallel  tests,  and  re- 
peated several  times. 

Furthermore,  symbiotic  and  antagonistic  effects  deserve  careful  con- 
sideration. Experiments  with  mixed  cultures  under  natural  conditions 


COUNTING,  CULTIVATING,  AND  TESTING  BACTERIA  77 

are  necessary  to  complete  and  to  confirm  pure  culture  studies.  In  the 
same  manner  as  the  medical  bacteriologist  combines  his  microscopical 
and  cultural  investigations  with  animal  tests,  so  it  is  necessary  that  the 
agricultural  bacteriologist  carry  his  work  from  the  laboratory  to  the 
dairy,  to  the  greenhouse,  and  to  the  field,  in  order  to  get  results  of  really 
scientific  as  well  as  of  practical  value. 


CHAPTER  VI 


STERILIZATION,  PASTEURIZATION,  ANTISEPSIS,  AND 

ASEPSIS 

At  present,  numerous  methods  are  available  for  eliminating  or  sup- 
pressing unwelcome  or  harmful  microorganisms.  Several  of  them  have 
been  used  since  ancient  times,  but  only  since  modern  bacteriology  has 
shed  light  upon  life  and  activities  of  bacteria  has  a more  rational  and 
successful  fight  against  these  minute  enemies  of  mankind  become  possible. 
Thorough  knowledge  of  their  properties,  especially  of  their  behavior  to- 
ward external  influences,  has  served  as  a basis  for  developing  the  modern 
methods  of  sanitation.  The  vast  majority  of  microorganisms,  however,  are 
not  harmful,  but  useful  to  mankind,  and  it  is  therefore  important  to  select 
in  each  case  the  proper  procedure  in  order  to  reach  the  desired  effect  with- 
out seriously  disturbing  the  action  of  useful  organisms. 

Effects  of  Various  Methods. — 'With  regard  to  the  more  or  less 
thorough  elimination  or  suppression  of  unwelcome  microorganisms  the 
methods  available  are  to  be  classed  as  follows : 

(a)  Sterilization  or  disinf ection,  aiming  at  the  complete  destruc- 

tion of  all  organisms  present.  The  term  disinfection  is 
used  as  a rule  when  special  attention  is  paid  to  the  killing  of 
pathogenic  (infective)  bacteria,  while  the  word  sterilization 
indicates  destruction  of  all  microorganisms  present.  But 
this  goal  is  not  always  reached ; the  high  resistance  of  bac- 
terial spores  makes  sterilization  often  incomplete. 

(b ) Pasteurization  and  antisepsis,  aiming  at  the  destruction  of  the 

majority  of  organisms  present,  especially  of  those  in  the 
vegetative  state.  Again  the  second  term  is  used  mostly  in 
medical  bacteriology,  while  pasteurization  is  of  more  gen- 
eral application.1 

1 The  term  antisepsis  is  derived  from  the  Greek  words  avrl  (anti-)  = against,  and 
afjfis  (sepsis)  = putrefaction,  decay.  The  term  pasteurization  was  introduced  to  honor 
the  memory  of  Louis  Pastern.  The  method  itself  was  known  before  Pasteur's  time; 
about  fifty  years  earlier  it  was  used  by  Appert,  by  the  great  French  chemist  Gay- 
Lussac,  and  by  others. 


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STERILIZATION , PASTEURIZATION  AND  ANTISEPSIS 


79 


(c)  Asepsis,  aiming  at  the  complete  exclusion  of  microorganisms 
and  prevention  of  development  of  those  present.  The 
meaning  of  the  term  is  that  no  putrefaction  takes  place.  In 
the  treatment  of  wounds  especially,  and  also  in  other  cases, 
asepsis  is  as  obviously  preferable  to  antisepsis,  as  is  pre- 
vention to  cure. 

The  treatment  directed  against  microorganisms  may  be  physical  or 
chemical  or  both  combined.  Always  the  relatively  most  efficient  pro- 
cedure must  be  selected  to  suit  the  individual  case;  there  is,  of  course, 
no  single  treatment  best  suited  to  all  cases.  The  question  of  greatest 
economy  requires  careful  consideration  in  this  connection. 

Most  radical  and  most  efficient  is  the  direct  application  of  the  open 
flame.  In  earlier  times  this  extreme  measure  had  often  to  be  relied  upon 
when  serious  epidemics  swept  the  countries.  Houses,  goods,  and  corpses 
had  to  be  destroyed  by  fire  in  order  to  check  the  disease.  Even  to-day 
such  a procedure  may  occasionally  become  necessary,  and  the  cremation 
of  bodies  appears  from  this  point  of  view  distinctly  superior  to  their 
interment  even  in  normal  times. 

With  all  other  less  thorough  methods  of  sterilization  and  pasteuriza- 
tion real  sterility  is  not  easily  attained.  But  even  if  all  germs  are  killed, 
their  bodies,  and  the  harmful  metabolic  products  which  they  may  have 
produced,  are  still  there,  and  the  very  wide-spread  assumption  that  milk, 
for  instance,  could  be  “freed”  from  all  detrimental  bacteria  by  thorough 
heating,  is  not  correct.  The  germs  are  merely  killed,  but  not  removed, 
and  their  products  may  still  be  active.  Perfectly  clean  milk,  aseptically 
handled,  is  much  more  preferable;  its  high  cost,  however,  prevents  its 
general  use. 

Physical  Treatment. — That  low  temperatures  merely  stop  fur- 
ther growth  and  action  of  bacteria,  but  exert  otherwise  very  little  effect 
upon  them,  has  been  emphasized  in  Chapter  IV,  2.  High  temperatures, 
on  the  other  hand,  are  of  fairly  satisfactory  effect,  especially  in  the 
presence  of  sufficient  moisture.  Moist  air  or  steam  is  always  much  more 
effective  than  dry  air  of  the  same  temperature.  Most  vegetative  cells 
are  killed  by  moist  heat  at  temperatures  ranging  from  50°  to  70°  C., 
more  slowly,  of  course,  at  the  lower,  more  rapidly  at  the  higher  tem- 
peratures. In  Swiss  cheese  factories  temperatures  of  50°  to  55°  C.  are 
applied  to  milk  and  curd  in  order  to  destroy  yeasts  and  other  organisms 
which  might  prove  harmful  if  they  were  allowed  to  grow.  In  haystacks 
and  manure  piles  partial  sterilization  by  spontaneous  heating  is  quite 
common.  Holding  milk  for  20  to  30  minutes  at  63°  C.  has  proved  to  be 
the  best  method  for  pasteurizing  market  milk,  because  about  99  per  cent 


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of  the  undesirable  organisms  are  killed,  while  flavor  and  other  qualities 
of  the  milk  are  not  much  impaired.  With  cream  (for  butter  making), 
as  well  as  with  skim  milk,  a short  time  exposure  at  85°  to  90°  C.  is 
preferable.  But  complete  sterilization  of  milk  is  an  extremely  difficult 
task  except  in  those  cases  where  resistant  spores  are  entirely  absent. 
More  than  104°  C.  can  not  be  applied  without  making  the  milk  unpala- 
table, and  despite  repeated  heatings  some  spores  may  survive,  which  will 
later  germinate  and  cause  spoilage  of  such  an  incompletely  sterilized 
product.  Complete  sterilization  is  assured  only  if  the  temperature  in  the 
autoclave  is  kept  for  one  hour  at  116°  to  120°  C.,  as  is  done  in  preserving 
meat  and  vegetables.  Nevertheless,  some  spores  may  survive  even  this 
severe  treatment,  although  more  frequently  spoilage  occurs  afterwards 
because  imperfect  containers  permit  new  contaminations. 

In  order  to  keep  the  germ  content  of  milk  low  from  the  start,  the  use 
of  sterilized  utensils  is  of  greatest  importance.  In  the  production  of 
certified  milk  this  point  requires  continual  attention.  In  America  steam 
treatment  is  still  often  applied  in  such  cases,  hut  investigations  made  in 
Europe  about  20  years  ago  have  definitely  shown  that  hot  air  treatment 
is  much  preferable.  The  moisture  remaining  in  steamed  vessels  facili- 
tates new  contaminations  and  new  growth  of  bacteria,  which  possibili- 
ties are  entirely  eliminated  in  the  other  case.  Glass  containers  withstand 
165°  C.  very  well,  which  temperature  is  usually  applied  in  bacteriological 
laboratories,  but  metal  utensils  are  better  kept  for  a longer  time  (about 
5 to  10  minutes)  at  a somewhat  lower  temperature  (130°  to  140°  C.). 

Drying  at  low  temperatures  kills  only  comparatively  few  of  the  vege- 
tative cells,  but  artificial  drying  at  high  temperatures  is  very  efficient. 
Dried  milk,  dried  potatoes,  etc.  are  practically  sterile  after  treatment, 
but  contamination  soon  sets  in  again. 

Several  investigators  have  tried  to  pasteurize  milk  by  exposing  it  to 
ultra-violet  rays;  the  results  were  quite  unsatisfactory.  But  an  analogous 
treatment  is  used  effectively  for  water  purification.  Several  French 
cities  have  adopted  it  for  their  water  supplies.1 

The  mechanical  elimination  of  bacteria  by  filtration  is  widely  used  for 
reducing  the  germ  content  of  water.  Natural  filtration  takes  place  in 
every  soil ; therefore,  water  from  deep  wells  is  practically  sterile.  Where 
surface  water  must  be  used  it  is  sent  through  special  filter  beds  con- 
structed of  sand  and  gravel  (Fig.  26).  Most  of  the  bacteria  are  retained 
by  the  layer  of  mud  which  accumulates  in  the  uppermost  part  of  the 
sand.  The  speed  with  which  the  water  passes  the  filter  must  be  care- 
fully regulated,  and  the  efficiency  of  the  filter  must  be  regularly  con- 
trolled by  bacteriological  tests. 

1 M , von  Recklinghausen,  Journ.  Franklin  Inst.,  vol.  17S,  1914,  p.  6S1. 


STERILIZATION,  PASTEURIZATION  AND  ANTISEPSIS 


81 


Fig.  26. — Filter  beds  (-^  nat.  size) 


Fig.  27. — Chamberland.  Fig.  28. — Berkefeld  filters  (\  nat.  size)  for 

Bougies  (|  nat.  size)  running  and  for  stored  water. 


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Complete  elimination  of  bacteria  and  other  microorganisms  from 
water  is  possible  by  the  use  of  special  bacteria  filters,  such  as  the  Cham- 
berland  filter,  shown  in  Fig.  27,  or  the  Berkefeld  filter,  as  seen  in  Fig.  28. 
The  filtering  is  done  through  so-called  bougies,  porous  cylinders  made 
from  porcelain,  asbestos,  infusorial  earth  (kieselguhr),  cellulose,  or  simi- 
lar materials,  closed  at  one  end,  with  pores  of  less  than  0.2 /j.  diameter. 
In  bacteriological  laboratories  such  filters  are  used  for  sterilizing  sub- 
strates which  do  not  stand  heating,  but  the  results  are  not  always  satis- 
factory. If  the  solutions  which  are  to  be  sterilized  contain  colloidal  sub- 
stances a smaller  or  larger  part  of  these  will  be  retained  by  the  filter  and 
will  clog  it  eventually.  Another  point  is  that  always  more  and  more 
organisms  are  found  to  be  able  to  pass  such  filters,  even  if  these  are  ab- 
solutely perfect,  that  is,  free  from  fine  cracks  and  holes  large  enough  to 
let  bacteria  of  average  size  pass  through.  Especially  among  the  disease 
producing  organisms  such  filterable  forms  are  well  known,  as  for  instance 
with  rabies,  foot  and  mouth  disease,  hog  cholera,  small-pox,  trachoma, 
infantile  paralysis,  typhus  fever,  Rocky  Mountain  spotted  fever,  etc.  But 
m addition  to  these,  probably  numerous  non-pathogenic  germs  are  filter- 
able, especially  many  of  the  gonidia  of  the  smaller  types  of  bacteria.  Fur- 
thermore, even  those  bacteria  which  because  of  their  size  can  not  pass  the 
pores,  have  often  shown  themselves  capable  of  growing  as  very  thin 
threads  through  the  filter,  if  these  are  continually  used  for  a long  time. 
This  possibility  is  to  be  kept  in  mind  if  such  filters  are  permanently  used 
in  the  household  in  the  belief  that  they  assure  a perfectly  safe  water 
supply,  which  they  do  not. 

Air  can  be  freed  from  bacteria  and  mold  spores  by  drawing  it  through 
sufficiently  thick  layers  of  cotton,  as  is  done,  for  instance,  in  certain  types 
of  milking  machines.  It  is  frequently  assumed  that  the  milk  itself  could 
by  freed  of  its  bacteria  by  sending  it  through  a cotton  filter.  But  com- 
paratively few  of  them  are  removed,  namely  those  clinging  to  the  dirt 
which  remains  on  the  cotton.  All  others  pass  through  the  filter,  because 
they  are  smaller  than  the  fat  globules  of  the  milk,  whose  diameters  vary 
usually  between  4 and  10,u. 

Simple  mechanical  removal  of  bacteria  by  scouring  and  scrubbing  is 
but  partly  successful,  especially  in  those  cases  where  the  bacteria  are 
resting  upon  a perfectly  smooth  surface.  But  any  irregularities  of  the 
surface,  even  if  they  are  hardly  visible  to  the  naked  eye,  may  afford  com- 
plete protection  to  the  bacteria.  Ordinary  cleanliness  is  therefore  by  no 
means  identical  with  bacteriological  cleanliness,  which  to  attain  requires 
a more  thorough  treatment. 

Chemical  Treatment. — Many  “antiseptic”  or  “disinfectant”  sub- 
stance are  known  at  present,  and  new  ones  are  recommended  nearly 


STERILIZATION,  PASTEURIZATION  AND  ANTISEPSIS 


83 


every  day.  They  are  very  helpful  in  the  fight  against  diseases,  but  their 
use  for  preserving  food  is  rightly  restricted.  The  ‘ ‘ pure  food  laws  ’ ’ prop- 
erly forbid  their  use  or  demand  that  their  presence  be  clearly  men- 
tioned. 

Whether  a chemical  substance  is  harmful  or  not  depends  mostly  on  its 
concent  ration.  Distinctly  poisonous  compounds  become  harmless  to  bac- 

teria if  present  in  very  small  quantities,  or  they  may  even  exert  a stimu- 
lating effect.  On  the  other  hand,  substances  which  normally  act  as 
nutrients,  may  become  harmful  if  consumed  in  too  large  quantities.  Fur- 
thermore, the  different  degrees  of  sensitiveness  of  the  various  microor- 
ganisms, and  especially  the  generally  high  resistance  of  their  resting 
forms  must  be  taken  into  account,  in  order  to  understand  correctly  why 
the  results  obtained  by  chemical  as  well  as  by  physical  treatments  may 
vary  widely  according  to  circumstances.  As  was  pointed  out  before, 
enzymes  are  usually  more  resistant  than  is  the  living  cell,  and  metabolic 
processes  may  therefore  still  proceed  after  all  living  cells  have  been 
killed  by  chemical  treatment ; in  other  words,  bacterial  propagation  is 
more  easily  suppressed  than  is  bacterial  activity. 

Among  the  various  substances  available  in  the  household  for  the 
“chemical  warfare”  against  microorganisms,  acids  are  often  used  advan- 
tageously because  the  majority  of  bacteria  are  very  sensitive  against  a 
distinctly  acid  reaction.  The  weakest  acid,  carbon  dioxide,  exerts  only  a 
slightly  retarding  effect,  as  was  pointed  out  on  p.  62.  Sauerkraut,  sour 
pickles,  and  silage  contain  usually  1 to  2 per  cent  lactic,  acetic,  and  other 
organic  acids,  which  suffice  to  stop  practically  all  bacterial  development ; 
but  molds  may  cause  spoilage  unless  their  growth  is  suppressed  by  the 
absence  of  air.  2 to  3 per  cent  mineral  acids,  like  sulfuric  and  hydro- 
chloric acids,  are  sometimes  used  for  treating  manure  from  diseased  ani- 
mals, or  for  sterilizing  wood  work,  rubber  parts,  etc.  The  antiseptic 
effect  of  different  acids  is  not  dependent  on  their  hydrogen  ion  concentra- 
tion, but  apparently  on  their  ability  to  penetrate  the  cell  wall.  Burri 
noticed,  for  instance,  the  following  relative  efficiencies  of  various  acids 
towards  several  milk  bacteria  d 


Hydrochloric  acid 

100 

Acetic  acid 

100 

Nitric  acid 

100 

Propionic  acid 

100 

Sulfuric  acid 

80 

Lactic  acid 

100 

Phosphoric  acid 

60 

Citric  acid 

40 

Formic  acid 

100 

Tartaric  acid 

20 

Basic  substances  must  be  applied  usually  in  higher  concentrations. 
Slaked  lime  proves  very  useful  in  stables  and  dairies.  In  the  soil,  too,  it 

1 Burri,  Landw.  Jahrb.  d.  Schweiz,  Bd.  26,  1912,  p.  475. 


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can  be  used  for  at  least  partial  sterilization,  if  applied  in  large  quantities. 
Still  more  efficient  than  lime  alone  is  a mixture  of  equal  parts  of  milk  of 
lime  and  of  a 20  per  cent  solution  of  caustic  soda.  2 to  5 per  cent 
sodium  carbonate  is  economical  and  very  active  if  applied  at  tempera- 
tures of  60  to  80°  C.  5 to  10  per  cent  caustic  potash  mixed  with  10 
per  cent  sodium  hypochlorite  makes  a very  strong  disinfectant,  known  as 
antif ormin.  Unfortunately,  the  tubercle  bacilli  are  resistant  against  all 
these  substances,  excepting  only  hot  sodium  carbonate  solution.  Anti- 
formin  is  used  to  separate  tubercle  bacilli  from  other  species  in  the  ex- 
amination of  sputum  and  other  pathological  material. 

Among  the  mineral  salts  mercuric  chlorid,  commonly  known  as  cor- 
rosive sublimate,  is  usually  considered  to  be  one  of  the  strongest  poisons. 
0.1  per  cent  often  suffices  to  kill  all  bacteria.  But  wherever  protein  sub- 
stances are  present,  an  insoluble  compound  is  formed,  and  the  effect  is 
greatly  reduced.  Copper  sulfate,  widely  used  for  controlling  plant 
diseases  caused  by  fungi,  may  also  be  advantageously  applied  to  check 
the  growth  of  algae  and  bacteria  in  water  reservoirs.  If  the  water  is  not 
rich  in  carbon  dioxide,  concentrations  as  low  as  1 part  per  million  have 
proved  effective.1  Chloride  of  lime  is  also  very  helpful  for  safeguarding 
the  water  supply  (in  most  cases  1 to  3 parts  per  million  will  suffice),  for 
reducing  the  germ  content  of  dairy  utensils,  and  as  a disinfectant  of 
manure  and  sewage.  Ammonium  fluoride  (0.5  per  cent)  is  especially 
suitable  for  the  sterilization  of  rubber  tubes.  Potassium  permanganate 
and  potassium  bichromate  are  also  efficient  in  relatively  low  con- 
centrations (t/2  to  1 per  cent).  Sodium  chloride,  on  the  other  hand,  be- 
comes active  only  in  very  high  concentrations,  and  the  effect  remains  in- 
complete. Even  25  per  cent  salt  does  not  exclude  all  growth  of  bacteria 
and  yeasts.2 

Besides  chloride  of  lime,  ozone  is  widely  used  for  water  sterilization. 
Many  European  cities,  as  Paris,  Nice,  Florence,  have  adopted  tins  method. 
Ozonization  of  milk  has  also  been  tried,  but  without  satisfactory  results. 
Somewhat  more  favorable  was  the  application  of  peroxide  of  hydrogen. 
In  Denmark  this  treatment  was  fairly  extensively  used  when  milk  was 
shipped  for  long  distances,  and  it  is  of  indisputable  value  for  preventing 
losses  in  times  of  milk  shortage.  It  retards  bacterial  growth  distinctly 
and  is  quite  harmless,  because  it  splits  up  into  water  and  oxygen,  but 
as  long  as  small  amounts  are  still  present  in  unchanged  state  the  taste  of 
the  milk  is  inferior.  Peroxide  of  hydrogen  is  of  greatest  value  for  the 
antiseptic  treatment  of  wounds. 

1 U.  S.  Dept.  Agr.,  Bur.  Plant  Industry  Bull.  64,  76,  100. 

2Wehmer,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  3,  1897,  p.  209;  F.  Lewandowsky, 
Archivf.  Hyg.,  vol.  49,  1904,  p.  47. 


STERILIZATION,  PASTEURIZATION  AND  ANTISEPSIS  85 

Best  known  among  the  organic  disinfectants  and  widely  used  are 
phenol  ( carbolic  acid ) and  related  compounds.  But  in  order  to  get  a full 
effect  a 5 per  cent  solution  must  be  applied,  preferably  at  about  40°  C. 
This  makes  such  disinfection  rather  expensive,  and  the  strong  odor  of 
these  substances  is  liable  to  act  unfavorably  upon  milk  and  other 
food  it  may  reach.  Under  various  trade  names  special  preparations  are 
sold  which  do  not  smell  quite  as  strong  as  does  carbolic  acid,  but  their 
pi  ice  is  usually  too  high.  Very  efficient  and  economical  is  formaldehyde, 
in  t/2  to  1 per  cent  concentration  useful  for  dairy  utensils,  rubber  tubes, 
etc.  In  combination  with  caustic  lime  or  permanganate  it  is  of  special 
value  for  the  fumigation  of  rooms  (5  g.  formaldehyde  per  cbm.  space). 
After  3 to  4 hours  the  remaining  formaldehyde  is  removed  by  adding 
ammonia  (3  g.  per  5 g.  formaldehyde).  High  humidity  of  the  air  and  a 
high  temperature  are  essential  for  securing  the  best  possible  results. 
Benzoic  and  salicylic  acids  are  not  infrequently  used  in  the  household  for 
protecting  food  against  spoilage.  Three-tenths  per  cent  is  an  efficient  and 
still  fairly  harmless  amount ; nevertheless,  complete  sterilization  by  heat 
is  preferable  to  the  use  of  any  chemical  substances. 

Combined  Treatment. — For  practical  purposes  the  simultaneous  ap- 
plication of  several  kinds  of  treatment  is,  of  course,  often  feasible  and 
desirable.  The  action  of  chemical  substances  can  be  considerably  in- 
creased by  a change  in  reaction,  as  well  as  by  an  increase  in  temperature. 
Ordinary  wash  suds  give  practically  complete  sterilization  if  they  are 
used  at  or  near  the  boiling  point.  Heat,  high  concentration,  and  acid 
reaction  combine  their  effects  in  the  sterilization  of  fruit  preserves.  High 
pressure,  which  otherwise  is  not  very  reliable,  has  proved  to  be  valuable 
for  the  treatment  of  acid  fruit  juices  j1  the  surviving  spores  can  not  ger- 
minate in  the  acid  medium.  Electrical  pasteurization  of  milk,  which  was 
tried  in  Liverpool,  England,  and  in  American  army  camps,2  acts  by  rais- 
ing the  temperature  to  about  70°  C.  and  perhaps  by  simultaneously  start- 
ing some  electrochemical  reactions  within  the  cells.  Meat  preserved  by 
smoke  is  at  the  same  time  dried  and  exposed  to  formaldehyde  and  other 
antiseptic  substances  in  the  smoke.  Salting  of  meat  and  fish  acts  by  in- 
creasing the  osmotic  pressure,  as  well  as  by  the  chemical  effect  of  sodium 
chloride  and  other  compounds  present  in  ordinary  salt.  Many  other 
combinations  of  physical  and  chemical  action  are  possible;  they  are  the 
most  practicable  means  of  preventing  detrimental  bacterial  action. 

1 Hite,  Biddings,  and  Weakley,  W.  Va.  Exp.  Sta.  Bull.  146,  1914. 

2 A.  K.  Anderson  and  R.  Finkelstein,  Jour.  Dairy  Science,  vol.  2,  1919,  p.  374. 


CHAPTER  VII 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS 

The  continuity  of  life  on  earth  is  dependent  upon  the  uninterrupted 
progression  of  the  cycle  of  matter,  as  was  discussed  on  p.  2.  There  is 
an  equilibrium  between  the  constructive  work  done  mostly  by  higher  or- 
ganisms, and  the  destructive  work  done  mostly  by  microorganisms ; the 
latter  have  been  properly  called  the  “mediators  between  death  and  life’’ 
of  higher  plants  and  animals.  Under  this  aspect  the  action  of  pathogenic 
bacteria  represents  a transgression  of  the  proper  domain  of  bacterial 
life,  because  it  accelerates  death  and  hastens  decomposition.  But  the 
activities  of  bacteria  and  related  microorganisms  are  by  no  means  always 
destructive,  as  those  of  the  higher  organisms  are  not  always  constructive. 
Large  amounts  of  the  organic  compounds  formed  by  the  latter  are  again 
destroyed  in  the  process  of  respiration;  certain  groups  of  bacteria,  on  the 
other  hand,  are  doing  eminently  constructive  work,  as  for  instance  the 
nitrogen-fixing  bacteria  in  the  root  nodules  of  leguminous  plants. 

Efficiency  and  Virulence. — As  was  emphasized  in  the  preceding 
chapters,  it  is  the  minute  size,  the  simple  but  effective  structure  of  the 
bacterial  cells,  and  their  capability  of  very  rapid  multiplication  under 
very  different  environmental  conditions  which  cause  their  stupendous 
efficiency.  One  or  a few  bacteria  are  practically  without  significance ; but 
if  suitable  circumstances  favor  their  multiplication  and  activity,  very 
soon  conspicuous  changes  will  result.  H.  W.  Conn  has  drawn  a very 
appropriate  comparison  between  bacteria  and  snowflakes ; singly  of 
nearly  no  weight  and  very  short-lived,  they  are  both  able  to  become  very 
powerful  if  present  in  very  large  masses.  Avalanches  destroy  men  and 
their  homes,  bacterial  epidemics  may  depopulate  human  settlements ; and 
enormous  economic  losses  are  caused  year  after  year  by  microorganisms 
liberating  nitrogen.  Fortunately,  however,  the  useful  activities  of  bac- 
teria, as  a rule,  exceed  their  detrimental  effects;  and  the  better  their 
properties  are  known  the  more  advantageous  use  can  be  made  of  them. 

Pathogenic  bacteria  act  in  most  cases  by  the  toxic  substances  they 
produce;  accordingly,  it  has  become  customary  to  speak  of  their  virulence 
in  order  to  refer  to  their  poisonous  properties.1  Unfortunately,  the  same 

1 Derived  from  the  Latin  word  virus  (plur.  vira)  =poison. 

83 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  87 


word  is  not  infrequently  used  for  non-pathogenic  bacteria,  too.  Several 
authors  have  written  upon  the  “virulence”  of  lactic  acid  bacteria,  of 
nitrogen  fixing,  and  of  other  useful  microorganisms,  where  they  meant,  of 
•course,  their  efficiency,  as  there  is  no  poison  produced  in  these  cases. 
Obviously,  the  words  efficient  and  virulent  can  be  used  synonymously  only 
for  pathogenic,  not  for  other  organisms.  Expressions  like  “virulence 
of  fermentation,  ’ ’ which  also  may  be  found  occasionally  in  the  literature, 
are,  of  course,  quite  incorrect  and  should  be  strictly  avoided. 

Relativity  of  Action. — Like  the  functions  of  all  other  living  organ- 
isms so  is  the  action  performed  by  microorganisms  always  dependent  on 
their  efficiency,  as  well  as  on  the  modifying  influences  of  outside  condi- 
tions. Good  seed  will  usually  produce  better  crops  than  can  be  expected 
from  poor  seed,  but  circumstances  may  arise  which  will  completely 
change  these  relations.  Good  seed  is  not  of  much  use  in  a badly  tilled 
poor  soil,  and  the  best  milk  cow  can  not  display  her  efficiency  if  proper 
feed  and  care  are  lacking.  The  situation  is  exactly  the  same  with  the 
lower  organisms.  Pure  cultures  of  the  highest  efficiency  could  be  selected 
and  cultivated  in  the  laboratory,  but  they  would  not  exert  any  appreci- 
able effect  under  unsuitable  conditions  in  the  dairy  or  in  the  soil.  There- 
fore, proper  environmental  conditions  are  no  less  important  than  is  the 
efficiency  of  the  organisms  concerned. 

But  outside  conditions  differ  and  vary,  as  does  the  efficiency  of  the 
bacteria.  Numerous  factors  exert  their  influences,  and  the  final  result 
is  determined  by  the  relations  existing  between  them.  The  great  varia- 
bility of  bacterial  cells  and  the  ability  of  most  microorganisms  to  adapt 
themselves  to  very  different  environmental  conditions  lead  to  changes 
in  bacterial  action  unknown  among  the  higher  organisms.  Such  char- 
acteristic functions  as  the  production  of  lactic  acid,  of  slime,  of  am- 
monia, and  the  fixation,  or  the  liberation  of  nitrogen  may  not  only  show 
wide  variations,  but  they  may  cease  entirely ; therefore,  in  the  laboratory 
great  attention  must  be  paid  to  keep  the  cultures  at  their  highest 
efficiency. 

Physical  and  Chemical  Actions. — Chemical  as  well  as  physical 
actions  take  part  in  the  transformation  of  matter  performed  by  the  lower 
as  well  as  by  the  higher  organisms.  From  a practical  standpoint  the 
metabolism  of  carbonaceous  and  nitrogenous  substances  is  undoubtedly 
of  greatest  importance.  The  chemical  changes  occurring  in  the  silo,  in 
ripening  cream  and  cheese,  in  rotting  manure,  and  in  the  soil  will  be 
discussed  on  the  following  pages.  But  in  connection  with  these  trans- 
formations various  physical  effects  of  bacterial  activity  are  to  be  ob- 
served, and  sometimes  these  physical  functions  appear  of  special  interest. 
This  holds  true  particularly  with  regard  to  the  production  of  color,  of 


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light,  and  of  heat  by  various  microorganisms,  which  will  be  briefly  con- 
sidered first. 

1.  PRODUCTION  OF  COLOR,  OF  LIGHT,  AND  OF  HEAT 

In  regard  to  the  physical  functions,  again  many  parallelisms  are 
noticeable  between  the  behavior  of  higher  and  of  lower  organisms.  Pig- 
mentation is  very  common  among  lower  and  higher  plants  and  animals ; 
numerous  light  producing  organisms,  foremost  fishes,  have  been  found 
especially  in  the  deep  sea;  fireflies  and  other  phosphorescent  insects  are 
well  known ; and  the  production  of  heat  is  noticeable  wherever  intensive 
respiration  takes  place. 

Pigment  Formation. — The  majority  of  microorganisms  do  not  dis- 
play any  pronounced  pigmentation.  Most  cultures  of  bacteria  and  yeasts 
appear  as  whitish  or  grayish,  soft,  slimy  or  dry  layers  covering  the  sub- 
strates, without  distinct  character  if  examined  with  the  naked  eye.  In 
Fig.  1,  Plate  Y,  this  whitish  growth  of  Bad.  coli  is  clearly  visible;  on 
potato  only  is  a brown  pigment  in  evidence.  Inspection  of  a yeast  cake 
will  demonstrate  the  analogous  appearance  of  a colorless  fungus.  Molds, 
too,  grow  mostly  without  color,  at  least  as  long  as  no  spores  are  formed. 
Their  white  network  of  threads  is  quite  common  on  sour  cream,  stale 
bread,  old  leather,  etc.  When  growing  in  liquids,  grayish  loose  flakes  are 
formed. 

If  colored  growth  appears,  the  pigment  either  remains  within  the 
cells  which  show  the  coloration,  or,  less  frequently,  it  leaves  the  cells  as  an 
excretion  which  diffuses  into  the  substrate,  as  may  be  seen  around  some 
of  the  colonies  visible  in  Fig.  1,  Plate  III.  On  the  other  hand,  some  molds 
as  well  as  bacteria  have  the  tendency  to  extract  certain  coloring  sub 
stances,  for  instance  Congo  red,  from  the  substrate  and  to  accumulate  it 
in  their  cells.  This  ability  is  sometimes  of  diagnostic  value,  and  it  also 
explains  why  with  several  molds  strains  of  different  coloration  occur, 
which  in  such  cases  is  merely  accidental.1 

Next  to  white  or  gray,  a yellow  pigmentation  is  most  frequent  among 
the  bacteria  as  among  flowers.  All  hues  from  pale  lemon  color  to  a 
deep  rich  orange  may  be  seen  (Plates  III  and  VI).  Pink  and  red  tints 
come  next  in  frequency.  A pink  yeast  and  Bad.  prodigiosum  are  shown 
on  Plates  III  and  V.  A bright  red  coloration  of  the  substrate  may  be  due 
to  the  growth  of  various  molds  or  of  Bad.  ery  thro  genes,  whose  yellow 
pigmented  cells  present  a striking  contrast  to  their  red  surrounding 
(Plate  VI).  Purple  colored  bacteria  occur  sometimes  in  very  large  num- 
bers in  the  water  of  ponds  and  ditches,  where  they  may  participate  in 


1 Marzinowsky,  Archi”  f.  Hyg.,  vol.  73,  1912,  p.  191. 


hand 


Lohnis-Fred,  Text  book 


Plate  V 


1.  Cultures  of  Bacterium  coli 

V,  nat.  size 


2.  Cultures  of  Bacterium  prodigiosum 

7 3 nat.  size 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  89 

the  oxidation  of  hydrogen  sulfide.  A soluble  fluorescent  substance,  pre- 
senting a green  or  bluish  green  color  in  reflected  light,  is  produced  by 
Bad.  fluorescens,  one  of  the  most  common  organisms  in  water,  milk, 
manure,  and  soil,  and  by  Bad.  pyocyaneum,  a species  connected  with  pus 
formation  in  wounds  and  sometimes  in  the  udder.  Bad.  fluorescens  is 
very  active  in  splitting  fats  and  in  producing  ammonia,  and  therefore  of 
special  interest.  A yellowish-green,  bluish-green,  or  grayish-green  pig- 
mentation is  frequently  noticeable  with  the  conidia  of  various  molds. 
Brown  and  black  colors  are  also  not  rare  with  lower  fungi  as  well  as 
with  bacteria.  Brown  or  black  varieties  of  Bad.  fluorescens  are  often 
met  with  in  stable  manure.  Some  strains  of  the  hay  and  potato  bacilli 
( Bac . subtilis  and  mesentericus)  are  also  able  to  produce  a black 
pigment,  which  is  very  characteristic  of  one  of  the  most  important 
nitrogen  fixing  species,  Azotobader  chroococcum.  Another  interesting 
group  of  soil  organisms,  usually  named  Actinomyces  chromogenes,  is 
characterized  by  a soluble  brown  pigment,  visible  around  one  colony  in 
Fig.  1,  Plate  III.  Blue  and  violet  colors  are  also  not  absent  among  bac- 
teria and  fungi.  Best  known  of  these  is  one  species,  called  Bad.  syn- 
cyaneum  or  B.  cyanogenes,  which  produces  in  slightly  acid  milk  an  in- 
tensive sky-blue  color  (Plate  YI)  ; occasionally  black  strains  occur  with 
this  species,  too. 

Practical  Importance  of  Pigment  Bacteria. — Before  the  milk  sepa- 
rators were  invented,  and  the  milk  had  to  be  kept  several  days  before  the 
cream  could  be  taken  off,  blue,  yellow,  red,  or  green  discolorations  of  the 
milk  were  rather  frequent.  Sometimes  they  proved  extremely  trouble- 
some, as  for  instance  in  the  case  which  ultimately  led  to  the  discovery 
of  B.  cyanogenes.  On  this  particular  farm  all  milk  turned  blue  during 
eleven  years,  and  the  owner  went  bankrupt  before  the  cause  was  dis- 
covered and  remedies  found.  Red  milk  was  and  is  also  not  rare. 
If  it  is  red  from  the  start,  admixture  of  blood  from  a diseased  udder  is 
the  cause ; but  if  the  color  appears  later,  almost  invariably  Bad.  erytliro- 
genes  is  responsible.  Repeatedly  Bad.  prodigiosum  has  been  blamed, 
but,  as  was  pointed  out  above,  this  species  produces  only  a faint  pink 
color  in  the  cream  of  its  milk  cultures,  which  fact  is  of  no  practical  con- 
sequence. Yellow  spots  on  cream  and  a greenish  discoloration  of  milk 
are  often  to  be  observed  if  milk  of  low  germ  content  is  kept  for  a long 
time  at  low  temperatures  (2  to  5°  C.). 

Bacterium  prodigiosum  has  received  its  name  the  “wonder  worker  ,,'L 
because  of  its  connection  with  the  sudden  appearance  of  red  spots  on 
bread,  meat,  and  especially  on  the  consecrated  wafers  kept  in  the  dark, 


1 The  Latin  word  prodigium  means  wonder. 


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damp,  medieval  churches  of  Europe.  Sometimes  these  “bleeding  hosts” 
were  merely  accepted  as  wonders  and  served  to  stimulate  religious  zeal, 
hut  in  other  cases  quite  innocent  people,  especially  Jews,  were  held  re- 
sponsible for  having  caused  these  “wounds  on  the  body  of  our  Lord”; 
and,  as  far  as  records  have  been  kept,  about  10,000  men  and  women  have 
lost  their  lives  due  to  fanatic  persecution  by  excited  ignorant  mobs. 

Changes  in  Pigmentation. — A few  anaerobic  bacteria  are  able  to 
produce  a red  color  in  the  absence  of  air.  In  all  other  cases  pigmentation 
ceases  under  such  conditions.  High  temperatures,  close  to  the  maximum 
endurable  by  the  various  pigment  producing  species,  give  also  colorless 
growths.  The  green  color  of  Bad.  fluorescens  appears,  as  a rule,  only  in 
media  of  alkaline  reaction ; the  sky-blue  pigment  of  B.  cyanogene s is 
deepened  by  acids,  as  was  said  before.  B.  prodigiosum  grows  yellowish- 
red  on  alkaline,  bluish-red  on  acid  substrates.  The  red  color  of  B.  ery- 
throgenes  is  formed  only  in  the  dark,  while  the  pigments  produced  by 
certain  molds  become  more  intense  under  the  influence  of  light.  Nutri- 
tion, especially  presence  or  absence  of  certain  salts,  plays  also  its  role  in 
this  case,  as  does  spontaneous  variability,  so  general  in  bacterial  life. 

White  strains  of  Bad.  prodigiosum  and  syncyaneum  are  very  fre- 
quent; they  should  certainly  not  be  classed  as  distinct  species,  as  was 
done  repeatedly.  It  is  still  less  appropriate  to  consider  small  differences 
in  the  tints  of  pigmentation  as  species  marks.  Natural  variability  and 
environmental  conditions  are  usually  responsible  for  such  differences. 
Changes  from  white  to  yellow  and  to  orange  are  very  frequent  among 
Micrococci.  Old  cultures  of  yellow  rods  often  turn  white;  on  the  other 
hand,  gradual  changes  are  known  to  occur  from  the  white  Bad.  coli  to 
yellow  rods.  Bad.  fluorescens  as  well  as  its  counterpart  Bad.  putidum, 
which  does  not  liquefy  gelatin,  but  is  otherwise  very  similar  to  the  first- 
named  organism,  display  their  parallelism  also  in  producing  brown  vari- 
eties, which  in  the  latter  case  can  not  be  sharply  distinguished  from  those 
of  Bad.  syncyaneum.  A close  relationship  between  these  species  is  very 
probable.  The  same  holds  true  concerning  a great  number  of  small  mo- 
tile liquefying  rods  producing  a red  pigment,  which  are  sometimes  classed 
as  separate  species,  although  there  is  much  greater  probability  that  they 
are  merely  varieties  of  Bad.  prodigiosum.  The  reddish  surface  growth 
characteristic  of  Camembert,  Brie,  and  of  some  other  kinds  of  French 
cheese  is  only  partly  due  to  the  presence  of  bacteria  and  molds  of  red 
color.  Some  white  and  yellowish  species  have  been  isolated  from  such 
material  which  produce  this  particular  coloration  by  symbiotic  action. 

Phosphorescence. — Production  of  light  by  plants  and  animals  is  of 
fairly  frequent  occurrence.  Molds  and  bacteria  again  act  along  the  same 
lines  as  do  the  higher  organisms.  The  phosphorescence  often  noticeable 


Lohnis-Fred,  Text  book 


Plate  VI 


i.  Milk  changed  by  pigment  bacteria 

Bacterium  Micrococcus  Bacterium  Bacterium  B.  fluorescens 

erythrogenes  aurantiacus  fluorescens  syncyaneum  var.  bruneum 


2.  Phosphorescent  bacteria  growing  on  fish 

photographed  in  their  own  light 
Va  nat.  size 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  91 


in  decaying  wood  is  caused  by  fungi,  while  that  of  seawater,  of  fish,  and 
of  other  food  is  produced  by  bacteria.  The  photograph  shown  on  Plate 
VI  was  made  in  the  darkroom  by  16  hours  exposure  to  the  dim  light  of 
the  phosphorescent  bacteria  forming  the  bright  patches  on  the  fish.  The 
light  produced  by  pure  cultures  is,  of  course,  somewhat  stronger,  and  it 
was  used  by  H.  Moliseh  and  others  for  making  interesting  photographic 
pictures  of  various  objects.1  This  investigator  has  also  recommended  the 
use  of  such  cultures  for  making  lamps  for  miners,  which  would  preclude 
any  danger  of  explosion.  Unfortunately,  the  phosphorescence  even  of 
very  active  cultures  is  rather  weak.  An  area  of  one  square  meter  cov- 
ered by  a good  growth  of  such  organisms  would  furnish  no  more  than 
1/1000  to  1/100  candle  power.2  Furthermore,  phosphorescence  is  very 
easily  lost  in  pure  cultures ; most  stock  cultures  of  this  kind  grow  well, 
but  do  not  emit  any  light.  It  becomes  evident  from  this  fact  that  this 
process  is  not  of  vital  importance.  The  oxidation  of  various  substances 
causes  the  phosphorescence,  which  can  be  produced  experimentally 
by  adding,  instead  of  bacteria,  oxidizing  chemicals  like  peroxide  of 
hydrogen,  bromine  water,  etc.  to  sterile  humus,  decayed  wood,  alkaline 
fish  broth,  and  similar  easily  oxidizable  substances.3 

Production  of  Heat. — Phosphorescent  bacteria  and  fungi,  like  fire- 
flies, are  mainly  objects  of  curiosity,  but  the  production  of  heat  by 
microorganisms  is  of  much  greater  interest  and  practical  importance. 
Combustion  of  various  substances  in  the  process  of  respiration  is  always 
the  cause  of  an  increase  in  temperature  with  the  lowest  as  well  as  with 
the  highest  organisms.  Wherever  large  amounts  of  organic  matter 
accumulate,  rapid  propagation  of  fungi  and  bacteria  will  take  place, 
which  participate  in  the  production  of  heat  simultaneously  with  the 
surviving  plant  cells  and  with  oxidizing  enzymes  present  in  the  material. 

If  the  water  content  is  very  high,  as  in  milk,  liquid  manure,  or  in 
other  solutions,  the  rise  in  temperature  is  hardly  noticeable.  In  ripening 
cream,  however,  where  the  percentage  of  water  is  somewhat  reduced,  an 
increase  of  1°  to  IV20  C.  is  usually  to  be  recorded.  A lower  content  of 
moisture  leads  to  such  well  known  results  as  are  obtained  with  fodder 
in  silos  and  with  stable  manure  in  hot-beds.  And  if  still  less  water  is 
present  the  temperature  may  reach  degrees  where  the  organic  matter 
assumes  more  and  more  the  character  of  coal  and  may  become  subject  to 
spontaneous  ignition. 

1H.  Molisch,  “Leuchtende  Pflanzen,”  2.  Aufl.,  1912. 

a Friedberger  und  Doepner,  Centralbl.  f.  Bakt.,  I.  Abt.  Orig.,  vol.  32,  1907,  p.  1; 
A.  Lode,  Centralbl.  f.  Bakt,  II.  Abt.,  vol.  22,  1909,  p.  421. 

3Weitlaner,  Verhandlungen  d.  Zoolog. -Botan.  Geselhchafl  zu  Wien,  vol.  61,  1911, 
p.  192;  Gerretsen,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  52.  1920,  p.  353. 


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Gradual  Increase  in  Temperature. — The  whole  process  can  be 
divided  for  the  sake  of  clearness  into  three  phases,  (1)  increase  in  tem- 
perature up  to  45°  C.,  (2)  from  45°  to  70°  C.,  (3)  above  70°  C.  The  first 
phase  is  to  be  accepted  as  a normal  occurrence,  the  second  one  is  of  use  in 
certain  cases,  the  third  one  is  always  abnormal  and  dangerous  on  account 
of  the  possibility  of  spontaneous  ignition.  Leaves,  green  fodder,  bran, 
hay,  tobacco,  etc.  enter  the  first  phase  quite  regularly  as  soon  as  suffi- 
ciently large  quantities  are  brought  together,  so  that  the  accumulating 
heat  can  not  be  dissipated  by  cooling  off  at  the  outside.  The  respiration 
of  living  plant  cells  produces  the  initial  heat ; later,  oxidation  caused  by 
enzymes  of  dying  and  dead  cells  comes  into  play,  supported  to  a vary- 
ing degree  by  the  respiration  of  bacteria  and  fungi.  In  some  cases, 
for  instance  in  curing  tobacco,  the  respiration  of  plant  cells  and  the 
activity  of  enzymes  alone  are  of  importance;  in  other  cases,  as  in  the 
rise  of  temperature  in  stored  peat,  bacteria  and  fungi  are  solely  respon- 
sible, but  as  a rule  all  three  factors  contribute  to  the  final  effect.  Under 
normal  conditions  the  rise  in  temperature  reaches  and  exceeds  40°  C. 
only  in  rare  cases.  Low  water  content,  as  in  hay  and  bran,  expulsion  of 
air  by  strong  pressure,  as  in  silage,  tend  to  keep  the  oxidation  within 
rather  narrow  limits. 

If  temperatures  of  40°  to  45°  C.  persist  for  several  days,  plant  cells  as 
well  as  most  of  the  common  microorganisms  will  die,  but  their  enzymes 
will  remain  active,  and  there  will  be  also  a new  growth  of  thermophilic 
bacteria,  actinomycetes,  and  fungi.  If  circumstances  are  favorable,  the 
temperature  will  continue  to  rise  until  70°  C.  is  reached.  Then  micro- 
organisms  as  well  as  enzymes  cease  to  work,  but  purely  chemical  reac- 
tions may  now  proceed  with  great  rapidity  in  the  hot  material.  Numer- 
ous so-called  species  of  thermophilic  bacteria  have  been  described  more 
or  less  incompletely,  and  it  is  not  to  be  doubted  that  their  number  could 
be  greatly  reduced  by  critical  tests.1  Most  of  them  produce  endospores 
which  guarantee  their  survival  during  periods  of  low  temperature,  when 
all  vegetative  cells  may  die.  Naturally,  also  the  thermophilic  organisms 
can  exert  their  influence  only  if  enough  oxygen,  oxidizable  matter,  and 
water  are  present.  In  material  like  coal  a rise  in  temperature  is  almost 
exclusively  caused  by  chemical  action,  as  for  instance  by  the  oxidation  of 
iron  sulfides. 

Spontaneous  Ignition. — If  the  temperature  reaches  70°  C.  and  rises 
above  this  point  the  danger  of  spontaneous  ignition  becomes  acute.  It 
goes  without  saying  that  this  last  phase  is  purely  chemical.  But  how  the 
ignition  finally  takes  place  is  still  a matter  of  dispute.  As  a result  of 

1 A review  of  the  thermophilic  organisms  was  given  by  Ambroz  in  CentraLbl.  f.  Bakl. 
I.  Abt.  Ref.,  vol.  48,  1910,  pp.  257,  289. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  93 


various  chemical  reactions  easily  oxidizable  substances  like  hydrogen, 
methane,  as  well  as  volatile  organic  substances  are  evolved,  whose  pres- 
ence is  indicated  by  the  peculiar  smell  of  such  hot  substances.  The  high 
temperature  increases,  of  course,  their  affinity  for  oxygen.  If  the  or- 
ganic matter  changes,  as  often  happens,  to  a porous  finely  divided  coal, 
these  volatile  substances  are  adsorbed  by  it  and  may  be  ignited  to  a 
slowly  proceeding  glow  whenever  oxygen  is  suddenly  admitted  in  large 
quantities  either  by  a strong  wind  or  by  the  removal  of  the  upper  and 
outer  layers.  Such  pyrophoric  (that  is  fire-bearing)  coal,  however,  was 
not  always  found  in  hay  stacks  which,  nevertheless,  did  show  spontaneous 
ignition.  It  seems  as  if  in  such  cases  the  volatile  oxidizable  products,  be- 
cause they  are  not  adsorbed,  accumulate  as  gases,  which  may  be 
blown  out  by  the  wind  or  may  escape  when  the  hay  is  taken  down.  In 
both  cases  the  ignition  is  very  sudden,  almost  like  an  explosion,  which 
fact  explains  why  this  type  of  spontaneous  ignition  has  been  very  little 
studied. 


2.  TRANSFORMATION  OF  ORGANIC  SUBSTANCES 

The  transformation  and  decomposition  of  products  and  residues 
formed  and  left  by  higher  plants  and  animals  represents  the  main  field 
of  bacterial  activity.  As  a result  of  physiological  and  biochemical  in- 
vestigations it  is  well  known  that  numerous  processes  participate  in  the 
formation  of  the  chemical  compounds  which  are  more  or  less  essential 
for  the  life  of  the  higher  organisms.  As  time  proceeds,  analogous  data 
will  accumulate  with  regard  to  bacterial  life.  At  present,  however,  only 
the  main  lines  have  been  studied  along  which  bacterial  activities  pro- 
ceed. But  enough  is  known  to  present  a fairly  complete  and  accurate 
survey  of  these  processes,  especially  as  far  as  they  are  of  importance  to 
the  agriculturist. 

From  daily  experience  it  is  well  known  that  many  volatile  sub- 
stances are  produced  by  bacteria  and  fungi  whose  chemical  composition  is 
rather  incompletely  known.  Agreeable  or  offensive  odors  and  flavors  can 
be  noticed  quite  generally,  and  frequently  the  nose  permits  the  formula- 
tion of  a more  correct  judgment  in  regard  to  the  presence  and  activity 
of  microorganisms,  than  does  the  eye  or  any  other  sense.  The  disagree- 
able smell  of  unclean  dairy  utensils,  for  instance,  indicates  their  con- 
dition very  clearly,  and  in  judging  milk,  butter,  and  cheese,  the 
determination  of  their  flavors  often  gives  more  reliable  results  than 
are  obtainable  by  chemical  methods. 

Putrefaction,  Decay,  Fermentation. — The  presence  or  absence  of 
offensive  odors  is  also  used  as  one  of  the  foremost  marks  for  making  a 


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simple  differentiation  between  the  various  modes  of  decomposition.  This 
was  the  case  especially  in  regard  to  the  terms  putrefaction  and  decay,  and 
in  books  written  some  decades  ago  many  details  about  putrid  odors  may 
be  found  which,  however,  did  not  lead  to  any  clear  insight  into  these  proc- 
esses. The  term  fermentation,  on  the  other  hand,  was  and  is  commonly 
applied  to  transformations  connected  with  a noticeable  evolution  of  gas. 
Since  bacterial  activities  are  better  known,  some  authors  have  advo- 
cated reserving  the  term  putrefaction  for  the  anaerobic  decomposition  of 
nitrogenous  substances,  the  word  decay  for  aerobic  nitrogen  transforma- 
tion, and  fermentation  for  the  destruction  of  carbonaceous  compounds. 
But  as  is  always  the  case  when  vague  popular  terms  are  introduced 
into  scientific  language,  the  new  meanings  attached  to  them  are  not 
generally  accepted,  and  misunderstandings  are  the  inevitable  results. 
Iiow  inconsistently  those  denominations  are  applied  is  clearly  demon- 
strated, for  instance,  by  the  use  of  expressions  like  alcoholic  fermentation 
and  urea  fermentation.  In  the  first  case  the  non-nitrogenous  end  product 
of  the  process  is  mentioned,  but  in  the  second  case  the  starting  point, 
which  is  here  a nitrogenous  substance,  is  used  for  designation.  Analogous 
terms,  like  acid  fermentation,  slimy  fermentation,  etc.,  are  by  no  means 
better ; and  it  is  undoubtedly  preferable  to  avoid  all  such  vague  expres- 
sions wherever  clear  scientific  terms  are  available. 

Nitrogen-Carbon  Ratio. — The  quantity  and  quality  of  nitrogenous 
and  non-nitrogenous  organic  compounds  are  of  foremost  importance 
among  the  factors  which  determine  the  general  course  of  bacterial  action. 
The  situation  is  very  similar  to  that  in  animal  feeding,  where  the  eco- 
nomic success  is  dependent  upon  the  proper  relation  between  proteins  and 
carbohydrates  in  the  food.  All  other  factors,  such  as  the  presence  or  ab- 
sence of  air,  the  degree  of  moisture,  the  reaction,  etc.,  will  also  exert  their 
influences,  but  it  is  the  effect  of  the  carbon-nitrogen  ratio  which  de- 
serves closest  attention.  If  there  are  comparatively  large  quantities  of 
carbohydrates,  as  in  silage,  manure,  and  milk,  the  metabolism  of  nitrogen 
takes  quite  another  course  than  in  those  cases  where  only  few  and  not 
easily  accessible  carbon  compounds  are  present,  as  in  soil.  It  depends 
pre-eminently  upon  the  carbon  supply  whether  nitrate  is  formed  by  bac- 
teria or  whether  it  is  destroyed,  whether  nitrogen  is  fixed  or  whether  it  is 
liberated  from  its  compounds.  These  facts  will  be  discussed  and  ex- 
plained on  the  following  pages. 

Enzymatic  Action. — Many  of  the  substances  which  are  attacked  by 
bacteria  and  fungi  are  insoluble  in  water,  and  it  is  therefore  self-evident 
that  their  transformation  must  be  due  to  enzymatic  action,  because  only 
soluble  substances  can  enter  the  bacterial  and  fungous  cells.  But  also  in 
the  case  of  soluble  substances  enzymes  are  known  to  be  of  importance,  and 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  95 


it  is  by  no  means  improbable  that  ultimately  all  bacterial  action  may  be 
found  to  be  caused  by  enzymes.  This  fact,  however,  should  not  be  mis- 
interpreted, as  is  sometimes  done,  by  asserting  that  the  enzymes  be  of 
greater  interest  and  importance  than  the  microorganisms  themselves. 
Enzymes  are  soluble  cell  products  specially  fitted  to  start  one  or  another 
chemical  transformation,  whose  products  are  usually  of  value  to  the  liv- 
ing cells.  The  latter  are  always  of  primary  importance,  because  they 
produce  the  enzymes.  The  digestive  processes  in  man  and  animal  are 
also  mostly  the  result  of  enzymatic  action,  but  nobody  will  contest  that 
this  is  only  of  secondary  importance  compared  with  the  primary  function 
of  the  living  organism. 

The  participation  of  enzymes  in  the  various  transformations  is  of 
great  significance  in  regard  to  the  intensity  and  long  duration  of  these 
processes.  A very  small  amount  of  rennet  is  sufficient  to  coagulate  large 
quantities  of  milk  (approximately  1:800,000)  and  the  enzyme  remains 
active  long  after  the  death  of  the  calf  from  which  it  was  taken.  Analo- 
gous relations  exist  between  bacteria  and  fungi  and  their  enzymatic  ac- 
tions. Certain  urea  bacteria,  for  instance,  produce  enzymes  which  con- 
vert in  one  hour  a quantity  of  urea  into  ammonia  which  is  more  than 
1000  times  heavier  than  the  weight  of  the  bacteria  themselves.  These 
enzymes  also  continue  to  act  long  after  the  bacteria  have  died,  and  the 
same  behavior  is  to  be  noted  with  the  enzymes  active  in  silage,  in  storage 
butter,  or  in  slow  ripening  cheese.  When  bacteria  first  start  growing, 
the  enzyme  production  is  weak.  It  increases  with  the  bacterial  develop- 
ment ; but  after  this  has  reached  its  height  and  the  cells  begin  to  die,  the 
enzymes  continue  to  accumulate.  The  result  is  that  the  maximum  in  the 
number  of  living  microorganisms  always  precedes  the  maximum  in  enzy- 
matic action.  This  relation  is  especially  marked  in  cases  where  the 
enzymes  remain  inside  of  the  living  cells  (so-called  endo-enzymes),  but  it 
is  also  noticeable  under  the  opposite  conditions,  that  is,  when  ecto- 
enzymes  are  produced. 

3.  THE  CYCLE  OF  NITROGEN 

Because  of  the  great  economic  importance  of  nitrogenous  compounds, 
their  transformations  have  been  thoroughly  studied  by  bacteriologists  as 
well  as  by  chemists.  Numerous  details  have  been  gathered  in  the  course 
of  these  investigations  which  will  be  discussed  later,  insofar  as  they  are 
of  general  interest.  At  present  the  fundamental  faces  concerning  the 
cycle  of  nitrogen  will  be  considered  in  order  to  get  an  accurate  view  of 
the  whole  subject. 

The  Phases  of  the  Cycle  of  Nitrogen. — Nitrates  and  ammonia  rep- 
resent the  sources  of  nitrogen  for  the  higher  plants,  which  transform 


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them  to  amino  acids,  amides,  and  proteins  by  combining  them  with  car- 
bonaceous material,  previously  prepared  from  carbon  dioxide  and  water. 
The  organic  nitrogenous  compounds  are  used  by  the  animals,  and  after- 
wards they  are  broken  up  and  returned  to  their  mineral  state  by  bacteria 
and  fungi.  But  microorganisms  participate  also  in  other  transforma- 
tions of  nitrogen,  of  which  eight  or  nine  are  fairly  well  known,  while 
some  others  are  still  more  or  less  in  doubt.  Figure  29  presents  in 
schematic  arrangement  all  these  possibilities;  full  drawn  lines  indicate 


Fig.  29. — Cycle  of  nitrogen.  (1)  Protein  decomposition,  (2)  Ammonia  formation, 
(3)  Nitrification,  (4)  Nitrate  reduction,  (5)  Assimilation  of  ammonia  and  amino- 
nitrogen,  (6)  Assimilation  of  nitrates,  (7)  Denitrification,  (8)  Ammonia  oxidation, 

(9)  Nitrogen  fixation. 

well  studied  transformations,  broken  lines  those  not  yet  adequately 
known.  Protein  decomposition  (1),  ammonia  formation  (2),  and  nitrifi- 
cation (3)  constitute  the  normal  steps  in  the  mineralization  of  nitrog- 
enous compounds  and  represent  the  counterpart  to  the  assimilation  of 
nitrogen  as  performed  by  the  higher  plants. 

Retrograde  changes  comprise  the  nitrate  reduction  (4)  from  nitrate 
to  nitrite  and  to  ammonia,  and  the  assimilation  of  amino,  ammonium 
and  nitrate  nitrogen  (5  and  6).  In  performing  these  changes  the  micro- 
organisms enter  into  competition  with  the  cultivated  plants,  and  may 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  97 

sometimes  become  distinctly  harmful  to  them.  The  change  from  nitrate 
to  protein  (6)  which  completes  the  cycle,  is  in  lower  as  well  as  in  higher 
plants  not  so  direct  as  indicated  in  Fig.  29.  It  is  very  probable  that  in 
all  these  cases  the  transformation  passes  through  the  ammonia  and 
amino  stages,  but  especially  with  regard  to  the  nitrate  assimilating  bac- 
teria and  fungi  no  details  concerning  the  chemistry  of  the  process  are 
known  at  present. 

Of  greater  importance  than  these  more  or  less  abnormal  retrograde 
changes  are  the  liberation  and  the  fixation  of  free  nitrogen.  The  libera- 
tion of  nitrogen  from  nitrate  or  nitrite,  known  as  denitrification  (7), 
seems  always  to  start  from  the  latter  compound ; but  the  possibility  re- 
mains that  elementary  nitrogen  is  also  directly  split  off  from  nitrates. 
The  oxidation  of  ammonia  (8)  to  free  nitrogen  and  water  is  another  still 
problematical  transformation,  which  seems  to  be  partly  responsible  for 
the  losses  of  elementary  nitrogen  from  barnyard  manure.  The  same 
holds  true  concerning  the  analogous  decomposition  of  amides  and  amino 
acids.  Nothing  definite  is  known  about  this  process,  but  there  are  indica- 
tions that  this  transformation,  too,  is  connected  with  the  escape  of  free 
nitrogen  from  the  manure  pile. 

The  fixation  of  free  nitrogen  (9)  leads,  as  far  as  is  known,  directly 
to  protein  compounds.  In  the  root  nodules  of  the  leguminous  plants,  as 
well  as  in  cultures  of  nitrogen  fixing  organisms  isolated  from  the  soil,  no 
other  products  of  assimilation  have  been  found.  But  it  is  very  probable 
that  amides  and  amino  acids  occur  as  intermediate  steps  in  these  as  in 
other  cases.  Some  authors  believe  that  there  are  also  bacteria  able  to 
unite  nitrogen  and  hydrogen  to  ammonia,  or  to  oxidize  nitrogen  directly 
to  nitric  and  nitrous  acids.  Well  founded  data  in  support  of  this  opinion 
are  not  yet  available;  but  in  view  of  the  fact  that  by  chemical  methods 
all  three  modes  of  nitrogen  fixation  can  be  performed,  the  possibility 
that  microorganisms  may  act  along  the  same  lines  should  not  be  a-priori 
rejected. 

Terminology.-— The  names  as  used  for  the  different  transformations 
of  nitrogen  are  mostly  applied  in  the  manner  just  described.  Sometimes 
the  term  “denitrification”  is  confounded  with  “nitrate  reduction,”  or 
even  with  “nitrate  assimilation,”  but  such  usage  is  not  to  be  recom- 
mended. If  merely  the  transformation  or  the  loss  of  nitrates  is  ascer- 
tained, but  not  investigated  which  one  of  the  three  processes  is 
actually  involved,  none  of  these  specific  terms  should  be  used,  but  simply 
“transformation”  or  “loss  of  nitrate.”  To  call  the  nitrogen  fixation 
“nitrification”  is  another  not  infrequent  mistake. 

Likewise  not  justified  is  the  use  of  the  term  “azofication”  or  “azoto- 
fication”  instead  of  nitrogen  fixation,  because  its  real  meaning  is  not 


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fixation,  but  “formation”  of  nitrogen,  analogous  to  ammonific-ation  = 
formation  of  ammonia,  and  nitrification  = formation  of  niter.  Quite  re- 
cently a new  term  “rhizofication”  was  proposed  1 to  designate  the  nitro- 
gen fixation  occurring  in  the  root  nodules  of  the  leguminous  plants.  It 
is  to  be  hoped  that  it  will  not  come  into  general  use,  because  it  is  very 
incorrectly  chosen;  it  means  “root  formation,”  not  nitrogen  fixation 
within  the  roots.2 

Decomposition  of  Protein  Substances. — On  account  of  the  varied 
compositions  of  the  proteins  their  decomposition  follows  many  different 
lines  and  leads  to  widely  differing  results.  A rapid  and  complete  min- 
eralization of  such  substances  is  desirable  in  manure  and  in  soil.  In 
other  cases,  for  instance  in  cheese  ripening,  only  a partial  change,  some 
kind  of  pre-digestion  of  the  proteins  is  attained  by  which  they  are 
partly  transformed  to  amides,  amino  acids,  and  ammonium  salts. 

Higher  organisms  participate  more  or  less  actively  in  the  first  phase  of 
the  nitrogen  cycle  in  direct  competition  with  bacteria  and  related  micro- 
organisms. The  proteins,  produced  by  the  plants  from  nitrate,  carbon 
dioxide,  and  water,  are  widely  used  and  transformed  by  the  higher  ani- 
mals. As  many  animals  live  on,  in,  and  from  another,  manifold  changes 
may  take  place  which,  however,  rarely  lead  below  the  first  step,  that  is, 
below  amides  and  amino  acids.  Close  competition  between  higher  and 
lower  organisms  is  noticeable,  for  instance,  in  the  intestines  as  well  as  in 
ripening  cheese,  provided  that  in  the  latter  case  mites  and  fly  larvae  are 
permitted  to  develop  on  its  surface. 

A multitude  of  protozoa,  molds,  yeasts,  and  bacteria  is  always  ready 
to  attack  any  protein  substances  not  immediately  used  by  higher  plants 
and  animals.  Aerobic  and  anaerobic,  psychrophilic  and  thermophilic 
microorganisms  may  become  active.  It  is  sometimes  asserted  in  the 
literature  that  “genuine”  putrefaction  is  caused  exclusively  by  anaerobic 
bacteria.  But  because  “genuine”  putrefaction  can  not  be  defined  ex- 
actly, this  statement  is  of  no  consequence.  It  is  to  be  admitted  that  cer- 
tain strains  of  anaerobic  bacteria,  belonging  to  the  group  of  Bac.  putrifi- 
cus,  produce  most  offensive,  putrid  odors.  However,  decomposition  of 
meat,  usually  accepted  as  an  example  of  true  putrefaction,  is  to  a large 
extent  due  to  the  activity  of  B.  proteus,  an  aerobic  organism.  Compara- 
tive tests  made  with  representatives  of  both  groups  of  bacteria  gave  the 
following  results  in  regard  to  the  lytic  actions  exerted  upon  the  protein 
nitrogen  within  one  month  at  37°  C. : 3 

1 P.  E.  Brown,  Jour.  Amer.  Soc.  Agron.,  vol.  13,  1921,  p.  323. 

2 Derived  from  the  Greek  word  f>Ua  (rhiza)  =root,  and  the  Latin  word  facere  = 
make. 

3H.  Tissier  Annal.  de  VInst.  Pasteur,  vol.  26,  1912,  p.  522. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  99 


Percentage  of  Nitrogen  made  soluble 

B.  putrificus 

B.  proteus 

Egg  albumin 

100 

2 

Blood  albumin 

86  to  91 

66 

Vegetable  proteins 

61  to  72 

45 

Meat 

82  to  100 

18 

Milk 

93  to  98 

26 

Lentils 

33  to  53 

3 

Many  other  aerobic  organisms,  common  in  manure  and  in  soil,  such  as 
B.  fluorescens  and  related  forms,  as  well  as  sporulating  bacteria  of  the 
subtilis-mesentericus  group,  may  participate  in  the  protein  decomposi- 
tion, especially  in  symbiosis  with  anaerobic  species  which  usually  display 
their  greatest  activities  in  the  initial  steps  of  the  process. 

Various  kinds  of  enzymes  are  active  in  these  transformations.  For 
attacking  insoluble  substances  ecto-enzymes  are  produced,  while  soluble 
substances,  able  to  enter  the  cell,  are  transformed  by  endo-enzymes. 
These  bacterial  enzymes  are  similar  to  those  found  in  higher  or- 
ganisms, especially  rennin,  pepsin,  trypsin,  and  erepsin,  all  active  in 
animal  digestion.  Cooperation  between  animal  or  plant  enzymes  and 
those  of  bacterial  origin  is  not  infrequent.  Milk  enzymes  and  rennet  com- 
bine their  effects  with  those  of  the  microorganisms  in  milk,  butter, 
and  cheese ; plant  enzymes  are  active  in  silage  together  with  bacteria 
and  fungi;  animal  enzymes  enter  the  manure  in  the  feces  along  with 
great  numbers  of  microorganisms. 

The  first  products  of  protein  metabolism  are  usually  of  great  nutri- 
tive value  and  are  therefore  repeatedly  used  by  higher  as  well  as  by 
lower  organisms.  Only  part  of  the  nitrogen  is  changed  into  amino  and 
ammonium  nitrogen,  and  this  delayed  and  incomplete  mineralization  is 
demonstrated  by  the  slow  and  moderate  fertilizing  effects  of  substances 
like  flesh  meal,  whale  guano,  barnyard  and  green  manures. 

Transformation  of  Amides  and  Amino  Acids. — Certain  amides  and 
amino  acids,  especially  asparagin,  aspartic  acid,  alanin,  leucin,  and 
tyrosin,  are  still  of  fairly  high  nutritive  value  for  numerous  microor- 
ganisms, as  was  demonstrated  for  asparagin  on  Plate  IV.  The  nitrogen 
present  in  such  form  is  therefore  not  readily  transformed  into  ammonia. 
This,  however,  is  the  case  with  those  amides  and  amino  acids  which  con- 
stitute the  nitrogenous  part  of  liquid  manure,  that  is  with  urea,  hippuric 
and  uric  acids. 

The  transformation  of  urea  into  ammonia  is  represented  by  the  follow- 
ing formula: 


CO(NH2)2+2  H20  = (NH4)2C03 


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Presence  or  absence  of  air  is  of  no  influence  upon  this  transformation. 
The  urea  bacteria  themselves  grow  better  under  aerobic  than  under 
anaerobic  conditions ; but  as  not  their  growth,  but  the  production  of  the 
active  enzyme  called  urease,  is  of  significance,  and  the  enzymatic  action 
is,  of  course,  independent  of  the  presence  of  oxygen,  rapid  transforma- 
tion of  the  urea  takes  place  in  liquid  manure  even  in  complete  absence  of 
air.  Therefore,  the  practical  usefulness  of  air-tight  covers  on  tanks  and 
pits  used  for  storing  liquid  manure,  is  not  to  be  explained  by  any  bene- 
ficial effect  Upon  the  amide  transformation  as  such,  but  merely  as  due  to 
the  protection  afforded  against  the  evaporation  of  ammonia.  Various 
cocci  and  bacilli  are  known  to  be  able  to  transform  urea  into  ammonia ; 
sometimes  they  are  grouped  into  special  genera,  Urobacillus,  Urococcus, 
and  Urosarcina.  But  their  activity  can  easily  cease,  and  they  are  in  fact 
not  representatives  of  separate  genera,  but  merely  varieties  of  such  com- 
mon species  as  B.  proteus,  coli,  prodigiosus,  fluorescens,  eryfhrogenes. 
There  are  a few  species  which  temporarily  may  display  a very  great  ac- 
tivity and  a very  pronounced  adaptation,  as  is  the  case  with  the  spore- 
forming Bacillus  ( Urobacillus ) Pasteuri,  but  they,  too,  can  live  without 
urea.  The  ability  to  produce  urease  is  not  restricted  to  bacteria.  Several 
fungi  as  well  as  higher  plants  follow  the  same  lines;  soy  beans,  for 
instance,  are  comparatively  rich  in  urease. 

The  transformation  of  hippuric  acid  is  generally  slower  than  that  of 
urea,  and  it  is  dependent  on  the  presence  of  oxygen  (in  air,  nitrate,  or  in 
sugar).  Glycin  and  benzoic  acid  appear  first,  according  to  the  formula 

C6H5CO.NHCH2COOH  +H20  = NH2CH2COOH +c6h5cooh 

Usually  the  same  organisms,  bacteria  as  well  as  fungi,  perform  this  and 
also  the  next  transformation  which  leads  to  ammonia.  They  are  in  part 
identical  with  those  which  hydrolyze  the  urea. 

Still  a little  more  complicated  and  less  rapid  is  the  transformation  of 
uric  acid  which  is  at  first  changed  to  allantoin  and  then  to  urea. 

I.  2 C5H4N403+02+2  H20  = 2 C02+2  C4H6N403 

II.  C4H6N403+02+H20  = 2 C02+2  CO(NH2)2 

Some  species  stop  at  urea,  while  others  continue  their  work ; B. 
fluorescens,  other  aerobic  short  rods,  and  several  molds  are  such  organ- 
isms. A sporulating  bacterial  species  named  Bac.  acidi  unci,  may  become 
active  under  anaerobic  conditions.1 II. 

The  transformation  of  cyanamid  passes  likewise  through  urea.  The 

1 Liebert,  Proc.  Acad.  Amsterdam,  vol.  17.,  1909. 


Lohnis-Fred,  Text  book  Plate  VII 


1.  Ammonia  production  and  bacterial  growth 

in  urea  broth  in  peptone  solution 


2.  Transformation  of  ammonia  and  nitrate 

in  soil  extract  containing  0.05  °/„  di=potassium  phosphate  and 
0.1  °/0  ammonium  sulfate-p^13^  or  0-1  °/o  nitrate  or  0.1  °/0  nitrate  -}- 1 °/0  sodium  citrate 


Nitrification 


Nitrate  assimilation.  Denitrification 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  101 

chemical  substance  contained  in  the  fertilizer  called  cyanamid  or  nitro- 
lime  is  calcium  cyanamid.  This  separates  quickly  in  moist  soil  into  cal- 
cium hydroxide  and  cyanamid.  The  latter  is  then  rapidly  changed  by 
soil  colloids  to  urea,  and  this  by  bacteria  to  ammonium  carbonate. 

I.  CN.NCa+2  H20  = CN.NH2+Ca(OH)2 

II.  CN.NH2+H20  = C0(NH2)2 

III .  CO  (NH2)  2 + 2 H20  = (NH4)  2C03 

No  bacteria,  but  some  fungi  are  known  to  be  able  to  attack  cyanamid 
directly;  under  natural  conditions,  however,  the  quicker  action  of  soil 
colloids  prevents  their  becoming  active.1  The  urea  derived  from 
cyanamid  is  not  transformed  by  the  typical  urea  bacteria,  but  by  various 
other  kinds,  which  seem  to  be  less  susceptible  to  the  poisonous  character 
of  the  intact  cyanamid  and  of  some  of  the  by-products  present  in  the 
commercial  fertilizer. 

Ammonia  Formation. — The  comparatively  simple  hydrolyzing  proc- 
esses occurring  in  liquid  manure  and  with  cyanamid  transfer  nearly  all 
nitrogen  into  ammonia,  and  only  very  little  of  it  is  used  for  sustaining 
the  life  of  the  enzyme  producing  microorganisms.  With  proteins  the 
opposite  relation  is  to  be  observed;  a luxuriant  growth  of  bacteria  and 
fungi  takes  place,  but  very  little  ammonia  is  liberated.  In  Fig.  1,  Plate 
VII,  two  culture  flasks  are  shown,  one  containing  beef  broth  to  which 
10  per  cent  urea  had  been  added,  the  other  filled  with  peptone  solution ; 
both  flasks  were  inoculated  witli  a few  drops  of  a manure  infusion.  The 
urea  broth  remained  clear,  no  bacterial  growth  is  visible,  whereas  the 
peptone  solution  is  turbid  and  covered  with  a grayish-yellowish  film.  But 
contrary  to  this  weak  or  luxuriant  development  of  bacteria,  much  am- 
monia was  produced  in  the  first  and  very  little  in  the  second  flask,  as  is 
indicated  by  the  strong  color  reaction  or  its  absence  on  the  strips  of 
turmeric  paper  placed  between  the  cotton  stopper  and  neck  of  the  flasks. 

The  cause  of  these  opposite  results  is  to  be  sought  in  both  the  nitro- 
genous and  carbonaceous  components  of  these  two  classes  of  nitrogen 
compounds.  The  proteins  are,  as  a rule,  good  sources  of  nitrogen  as  well 
as  of  carbon,  while  most  of  the  amides  and  amino  acids  are  deficient  in 
one  or  the  other  direction  or  in  both.  In  the  presence  of  large  quantities 
of  easily  accessible  carbonaceous  compounds,  such  as  carbohydrates, 
glycerol,  and  similar  substances,  those  amides  and  amino  acids  are  also 
very  readily  assimilated  by  numerous  bacteria  and  fungi,  and  the  for- 
mation of  ammonia  is  much  reduced. 


1 Lohnis,  Zeitschr.f.  Garungsphysiologie,  vol.  5,  1914,  p.  16. 


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Ultimately,  however,  all  organic  nitrogen  will  be  ammonified,  and 
even  such  resistant  or  poisonous  substances  as  chitin,  quinine,  strychnin, 
morphin  and  nicotin,  or  the  toxins  produced  by  pathogenic  bacteria  are 
no  exceptions  to  this  rule.1  The  regular  course  of  the  nitrogen  cycle  may 
be  delayed,  but  it  will  never  be  broken.  As  was  mentioned  above,  very 
numerous  species  are  collectively  active  under  anaerobic  and  under  aerobic 
conditions,  at  high  as  well  as  at  low  temperatures.  Ammonification  is 
still  noticeable,  for  instance,  in  the  soil  when  its  temperature  is  close  to 
the  freezing  point. 

Nitrification. — The  transformation  of  ammonia  into  nitrite  and 
nitrate  represents  the  last  step  in  the  mineralization  of  nitrogen  com- 
pounds. Two  groups  of  bacteria  participate  in  this  process.  At  first  the 
nitrite  bacteria  become  active  according  to  the  following  formula : 

2 NH3+3  02  = 2 HNO2+2  H20 
Then  the  nitrate  bacteria  complete  the  oxidation: 

2 HN02+02=2  HN03 

Lime  and  other  basic  substances  of  the  soil  neutralize  the  acids  formed. 
Whether  or  not  organisms  exist  which  are  able  to  oxidize  ammonia,  or 
perhaps  even  organic  nitrogenous  compounds,  directly  to  nitrate,  is  not 
known  at  present.  Some  authors  have  advanced  such  opinions,  but  no 
convincing  proof  has  been  furnished.  The  laboratory  air  contains,  as  a 
rule,  small  amounts  of  nitrous  acid  which  are  readily  absorbed  by  slightly 
alkaline  solutions  if  these  are  kept  for  a few  weeks.  Therefore  a little 
nitrite  is  to  be  found  in  nearly  every  liquid  culture,  but  this  should  not 
be  accepted  as  valid  proof  of  nitrite  formation. 

Pure  cultures  of  nitrifying  bacteria  were  first  obtained  by  the  Kussian 
bacteriologist  S.  Winogradsky  in  1890;  but  the  existence  of  the  two 
groups  of  organisms  was  known  before  that  time,  and  numerous  data  in 
regard  to  their  behavior  had  been  collected  in  earlier  years  by  Sehlosing, 
Muntz,  and  Deherain  in  France,  and  by  Warington  and  Frankland  in 
England.  One  of  the  peculiarities  of  the  nitrifying  bacteria  is  their  great 
sensitiveness  to  large  quantities  of  soluble  organic  substances.  Thein- 

1 Concerning  the  ammonification  of  chitin  see  W.  Benecke,  Bot.  Zeitg.,  I.  Abt. 
vol.  63,  1905,  p.  227,  and  K.  Stormer,  Jahresber.  d.  Ver.f.  nngew.  Bot.,  vol.  5,  1907; 
p.  128,  concerning  quinine,  morphin,  strychnin,  Soyka,  Arch.f.  Hyg.,  vol.  2,  1884,  p.  281; 
concerning  nicotin,  .1.  Behrens,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  7,  1901,  p.  1;  concerning 
bacterial  toxins,  Charrin  et,  Mangin,  Cnmpt.  rend.  Soc.  Biol.,  vol.  49,  1S97,  p.  545, 
and  E.  Metchnikoff,  1.  c.,  p.  592. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  103 


organic  respiration  supplies  them  with  energy  for  assimilating  carbon  di- 
oxide, and  like  the  green  plants  they  are  unable  to  grow  in  substrates  con- 
taining considerable  quantities  of  carbohydrates,  organic  salts,  or  organic 
nitrogenous  compounds.  Gelatin  and  ordinary  agar,  therefore,  do  not 
permit  growth,  and  platings  could  be  made  successfully  only  after  W. 
Kiihne  had  introduced  silica  jelly  for  such  purposes.  Later  it  was  shown 
by  Beijerinck  that  agar  can  also  be  used  after  it  is  freed  from  all  soluble 
material  by  careful  washing. 

The  nitrite  bacteria  grow  either  as  small,  oval,  motile  rods  with 
polar  flagella  (Fig.  5,  Plate  II),  or  in  larger,  immotile,  globular  form. 
They  were  named  by  Winogradsky  Nitrosomonas  and  Nitrosococcus, 
respectively,  but  it  is  very  probable  that  the  coccoid  type  represents 
merely  the  growth  of  regenerative  bodies  of  Nitrosomonas.  The  nitrate 
bacteria  are  short,  immotile  rods,  which  have  received  the  name 
Nitrobacter.  Investigations  upon  their  sensitiveness  to  organic  sub- 
stances and  ammonia  were  made  by  Winogradsky  in  cooperation  with 
Omelianski.1  The  following  quantities  were  found  to  inhibit  growth  and 
action  of  the  nitrifying  organisms  in  alkaline  solutions : 

Pepton  Asparagin  Urea  Glucose  Ammonia 
Per  Cent  Per  Cent  Per  Cent  Per  Cent  Per  Cent 


Nitrosomonas 0.2  0.3  ? 0.2  — 

Nitrobacter 1.25  0.5  to  1.0  1 0.2  to  0.3  0.015 


Winogradsky  drew  from  these  findings  two  conclusions  which  were  gen- 
erally accepted  and  widely  copied  in  bacteriological  textbooks.  They 
read:  All  soluble  organic  substances  must  be  decomposed  in  the  soil 
before  nitrification  can  take  place,  and  all  ammonia  must  be  first  con- 
verted into  nitrite  before  Nitrobacter  can  begin  its  activity.  However, 
these  generalizations  were  not  supported  by  the  facts  observed.2  Under 
normal  conditions  the  soil  solution  is  nearly  neutral  and  does  not  contain 
such  large  quantities  of  soluble  organic  substances  as  were  found  to  be 
detrimental,  and  Nitrobacter  is  very  sensitive  only  to  free  ammonia, 
but  not  to  such  ammonia  salts  as  are  present  in  the  soil.  Nitrite 
and  nitrate  formation  proceed,  as  a rule,  simultaneously  in  the  soil,  and 
the  organic  substances  of  the  soil,  that  is,  humus  compounds,  are  not 
detrimental  but  usually  very  favorable  to  the  nitrifying  organisms,  so 
that  in  most  cases  the  more  humus  is  present  in  a soil  the  more  active  are 
its  organisms.  Highly  acid  peat  soils  are  to  be  excepted,  of  course,  not 
so  much  on  account  of  their  organic  substances,  as  because  of  the  fact 

1 Winogradsky  und  Omelianski,  Centralbl.  f . Bakt.,  II.  Abt.,  vol.  5,  1899,  p.  436. 

2 Lohnis,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  13,  1904,  p.  706;  Fred  and  Davenport, 
Soil  Science,  vol.  11,  1921,  p.  389. 


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that  they  do  not  contain  a sufficient  amount  of  basic  compounds  neces- 
sary to  neutralize  the  nitric  and  nitrous  acids,  and  not  enough  oxygen  to 
permit  rapid  oxidation. 

In  barnyard  manure,  as  well  as  in  liquid  manure,  only  little  or  no 
nitrification  takes  place,  because  the  presence  of  soluble  organic  sub- 
stances and  the  restricted  supply  of  air  both  exert  their  adverse  in- 
fluences. But  if  stable  manure  is  exposed  to  the  air  in  thin  layers  a 
marked  nitrification  may  establish  itself.1 

Some  authors  have  thought  that  the  nitrification  of  ammonia  be  regu- 
larly connected  with  losses  in  free  nitrogen,  usually  estimated  at  about 
10  per  cent.  If  unfavorable  conditions  prevail,  and  therefore  ammonium 
nitrite  can  accumulate  in  considerable  quantities,  such  losses  are  indeed 
possible  according  to  the  formula : 

(NH4)N02  = 2 H20+N2 

Usually,  however,  this  reaction  is  of  no  importance,  and  it  has  been 
repeatedly  ascertained  in  exact  experiments  that  the  nitrification  as  such 
does  not  entail  any  losses.  The  nitrogen  requirements  of  the  nitrifying 
organisms  are  so  low  that  for  all  practical  purposes  it  can  be  assumed 
that  100  parts  of  ammonium  nitrogen  will  give  100  parts  of  nitrate 
nitrogen,  provided  that  this  transformation  is  not  disturbed  by 
antagonistic  actions. 

Nitrate  Reduction. — While  nitrification  is  the  work  of  probably  not 
more  than  two  groups  of  highly  specialized  organisms,  there  is,  on  the 
other  hand,  a great  number  of  bacteria  and  fungi  capable  of  causing  the 
opposite  reaction.  Some  of  them  reduce  the  nitrate  only  to  nitrite,  others 
confine  their  action  to  the  reduction  of  nitrite  to  ammonia,  but  most  of 
them  perform  the  complete  retrograde  transformation  from  nitrate  to 
ammonia.  This  function,  however,  is  rather  inconstant  and  may  be 
present  or  absent  among  closely  related  varieties  of  one  species.  It  can 
therefore  not  be  used  for  diagnostic  purposes. 

Easily  oxidizable  substances  of  the  soil,  first  of  all  humus  compounds, 
may  participate  actively  in  the  nitrate  reduction.  In  peat  soils  it  some- 
times happens  that  the  biological  nitrate  reduction  is  completely  replaced 
by  this  purely  chemical  reaction.  But  even  if  microorganisms  are  active, 
it  is  not  always  their  need  of  oxygen  which  is  responsible  for  the  reduc- 
tion. Frequently  products  of  their  metabolism  are  easily  oxidized  and 
liable  to  reduce  the  nitrate.  Accordingly,  the  transformation  of  nitrate 
to  nitrite  often  takes  place  in  the  presence  of  air,  but  the  second  step  from 
nitrite  to  ammonia  requires  always  more  or  less  anaerobic  conditions. 

1 Niklewski,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  26,  1910,  p.  38S. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  105 


These  facts  explain  why  nitrate  reduction  plays  a conspicuous  role 
only  in  peaty,  swampy,  water  logged  soils.  Heavy  rains,  inundations,  and 
insufficient  drainage  may  establish  similar  conditions  in  other  soils  too, 
but  in  general  the  nitrate  reduction  is  not  of  very  great  importance.  As 
soon  as  air  again  pervades  the  soil  the  ammonia  is  once  more  quickly 
nitrified. 

Useful  application  of  nitrate  reduction  is  made  in  the  cheese  industry 
when  saltpeter  is  added  to  the  curd  in  order  to  prevent  gassiness  of  the 
cheese  produced.  If  no  nitrate  is  added  the  possibility  exists  that  part 
of  the  lactose  will  be  used  by  certain  bacteria  as  an  oxygen  supply,  and 
the  liberated  hydrogen  and  carbon  dioxide  will  cause  the  deformation  of 
the  cheese.  Nitrate,  however,  prevents  this  fermentation  by  serving  as 
an  easily  accessible  source  of  oxygen ; the  ammonia  formed  is  promptly 
changed  into  harmless  organic  salts. 

Assimilation  of  Amino,  Ammonium,  and  Nitrate  Nitrogen. — The 
presence  or  absence  of  oxygen  determines  whether  nitrification  or  nitrate 
reduction  will  take  place,  and  it  is  the  absence  or  the  presence  of  large 
amounts  of  easily  accessible  organic  carbon  compounds  which  decides 
whether  ammonia  will  be  formed  and  nitrified,  or  whether  microorgan- 
isms will  assimilate  nitrate,  ammonium,  and  amino  nitrogen.  Because 
soluble  organic  compounds  are  very  common  in  nature,  it  is  self-evident 
that  this  assimilation  of  the  simpler  nitrogen  compounds  is  no  rare  oc- 
currence, and  therefore  of  greater  importance  than  the  nitrate  reduction. 
It  was  pointed  out  before  (p.  43  and  Plate  IV)  that  a relatively  poor 
source  of  nitrogen,  such  as  urea,  ammonia,  and  nitrate,  becomes  accessible 
to  most  of  the  microorganisms  only  if  a good  source  of  carbon  is  simul- 
taneously present.  Urea  is  quickly  and  completely  transformed  into 
ammonia  in  soil  where  very  little  soluble  organic  substances  are  to  be 
found,  but  in  barnyard  manure  30  to  70  per  cent  of  the  urea  nitrogen  is 
assimilated  and  transformed  into  bacterial  proteins.  If  a large  quantity 
of  straw  or  of  fresh  manure  is  plowed  under  a few  days  or  weeks  before 
a new  crop  is  planted,  it  will  not  show  any  marked  fertilizing,  but  often 
a distinctly  disadvantageous,  effect,  because  the  carbohydrates  and 
organic  salts  contained  therein  will  enable  numerous  microorganisms  to 
assimilate  ammonium  and  nitrates,  previously  formed  in  the  soil. 

The  assimilation  of  amino,  ammonium,  and  nitrate  nitrogen  is  per- 
formed by  aerobic  organisms.  As  a rule,  the  nitrogen  of  amides  and 
amino  acids  is  more  readily  utilized  than  that  of  ammonium  salts,  and  this 
is  generally  better  assimilated  than  nitrate  nitrogen.  Under  average  soil 
conditions  very  little  nitrate  nitrogen  becomes  inaccessible  to  the  roots  of 
the  higher  plants  on  account  of  the  interference  of  nitrate  assimilating 
bacteria  and  fungi.  But  almost  without  exception  a more  or  less  marked 


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assimilation  of  ammonium  salts  takes  place  in  all  soils,  even  if  no  fresh 
manure  or  straw  has  been  added.  The  fertilizing  effect  of  ammonium 
sulfate  is  therefore  frequently  more  or  less  inferior  to  that  of  sodium 
nitrate.  Especially  in  light  soils,  where  the  physical  and  chemical  ab- 
sorption of  ammonia  is  not  very  strong,  sometimes  the  biologic  fixation 
of  ammonia  by  assimilating  microorganisms  may  become  very  marked. 

Repeatedly  the  statement  has  been  made  in  the  literature  that  the 
assimilation  of  ammonium  nitrogen  is  done  mostly  by  molds,  while  that 
of  nitrate  nitrogen  is  declared  to  be  due  to  bacterial  activity.  This  gen- 
eralization, however,  is  not  tenable.  Whether  fungi  or  bacteria  will  pre- 
dominate depends  mostly  on  the  source  of  carbon  and  on  the  reaction  of 
the  substrate.1  Ammonium  carbonate,  for  instance,  gives  only  bacterial 
growth.  Organic  ammonium  salts,  but  also  nitrate,  are  assimilated 
mostly  by  molds  if  glucose  is  present,  because  this  is  easily  converted  into 
acids.  Sulfates  and  chlorides  of  ammonium  stimulate  the  development  of 
fungous  growth,  because  they  are  physiologically  acid,  that  is,  the  acids 
are  left  and  make  the  substrate  sour  when  the  ammonium  is  used.  But 
in  the  presence  of  certain  carbon  compounds  bacteria,  too,  may  display 
vigorous  growth,  as  was  shown  on  Plate  IY  in  regard  to  ammonium  sul- 
fate and  glycerol. 

Whether  the  assimilation  is  done  by  bacteria  or  by  molds  is  of  con- 
siderable importance,  because  the  renewed  mineralization  of  the  con- 
verted nitrogen  proceeds,  as  a rule,  fairly  rapidly  if  bacterial  cells  and 
their  products  are  present,  whereas  spores  and  conidia  of  molds  are  much 
more  resistant.  Young  spore-free  mycelia,  however,  behave  like  bacteria. 
Comparative  tests  have  shown2  that  20  to  40  per  cent  of  such  bacterial 
nitrogen  was  nitrified  in  soil,  where  at  the  same  time  and  under 
analogous  conditions  only  4 to  8 per  cent  was  mineralized  if  mold  growth, 
rich  in  spores,  was  used  as  source  of  nitrogen.  This  slow  and  incomplete 
mineralization  of  bacterial  and  fungous  cells  makes  the  assimilation  of 
amino,  ammonium,  and  nitrate  nitrogen  by  microorganisms  a distinctly 
disadvantageous  process,  which  should  be  avoided  as  far  as  possible. 
Proper  rotting  of  stable  manure  and  of  straw  before  their  incorporation 
into  the  soil  is  helpful  in  this  respect. 

Liberation  of  Nitrogen. — Losses  of  nitrogen  by  purely  chemical  re- 
actions may  occur,  if  amides  and  ammonia  have  an  opportunity  to  react 
with  free  nitrous  acid  according  to  the  formulae : 

CO(NH2)2+2  HN02  = 2 N2+3  H20+C02 
NH3 + HN 02 = N 2+ 2 HoO 

1 St.  Bierema,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  23,  1909,  p.  672. 

2 Bierema,  1.  c. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  107 


These  processes  play  their  roles  probably  in  the  manure  pile,  as  well  as 
in  certain  peat  soils,  where  as  a result  of  improper  handling  (excessive 
liming,  especially)  considerable  quantities  of  the  peat  nitrogen  are  some- 
times transformed  into  ammonium  nitrite  and  more  or  less  completely 
liberated.1  Very  large  applications  of  ammonium  sulfate  may  cause 
similar  losses  in  normal  soils,  too,  as  was  demonstrated  by  Th.  Schlosing,2 
who  found  that  4 to  8 per  cent  nitrogen  disappeared  when  ammonium 
sulfate  was  used  in  quantities  equivalent  to  4000  to  7000  lbs.  N per  acre, 
that  is,  about  100  times  as  much  as  is  applied  normally. 

Among  the  other  ways  in  which  nitrogen  may  be  liberated,  the  decom- 
position of  amides  and  of  ammonia  seems  to  be  of  importance  especially 
in  regard  to  the  transformation  of  nitrogen  in  barnyard  manure.  But, 
as  was  pointed  out  above,  no  definite  data  in  these  respects  are  available 
at  present.  The  only  well-known  mode  of  liberation  of  nitrogen  by  bac- 
teria is  the  so-called  dentrification,  that  is,  the  decomposition  of  nitrates 
and  nitrites  under  anaerobic  conditions  with  liberation  of  free  nitrogen. 

Denitrification. — As  is  the  case  with  nitrate  reduction,  there  are  two 
possibilities  of  denitrification.  Either  a “direct”  denitrification  is  per- 
formed by  bacteria  and  their  enzymes,  or  an  “indirect”  denitrification 
takes  place,  caused  by  hydrogen  and  other  easily  oxidizable  substances 
resulting  from  the  decomposition  of  organic  substances.  Furthermore, 
certain  sulfur  bacteria  may  act  as  denitrifiers ; but  this  is  a rather  rare 
case  and  therefore  not  of  general  interest.  It  will  be  explained  in 
Chapter  VII,  5. 

Nearly  all  the  nitrogen  split  off  by  direct  denitrification  appears  in  the 
free,  elementary  form,  while  indirect  denitrification  gives  rise  to  smaller 
or  larger  quantities  of  nitric  oxide  (N202)  and  nitrous  oxide  (N,0)  be- 
sides free  nitrogen.  Naturally  both  processes  occur  often  simultaneously. 
The  indirect  denitrification  is,  of  course,  of  no  importance  to  the  organisms 
whose  metabolic  products  are  oxidized,  but  the  direct  denitrification 
enables  otherwise  aerobic  bacteria  to  live  under  strictly  anaerobic  con- 
ditions. The  oxygen  taken  from  nitrate,  nitrite,  and  from  nitrous  oxide 
replaces  the  free  oxygen  in  the  process  of  respiration,  according  to  the 
following  equations : 

2 KNO3+2  C. . =N20+K2C03+C02 
2 KN02  + C..  =N20+K2C03 
2 N20  + C. . =2  N2+C02 

1 Th.  Arnd,  Landw.  Jahrb.,  vol.  47,  1914,  p.  371. 

2Th.  Schlosing,  Compt.  rend.  Acad.  Paris,  vol.  109,  1S89,  p.  884. 


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Nitrite  and  nitrous  oxide  are  frequently,  though  not  always,  traceable  as 
intermediate  products. 

The  number  of  denitrifying  bacteria  described  thus  far  is  rather 
large,  but  it  is  beyond  doubt  that  many  of  these  so-called  species  are 
merely  varieties  of  other  species  which  usually  do  not  denitrify.  Various 
authors  baptized  their  respective  cultures  Bacterium  or  Bacillus  denit ri- 
ftcans  with  or  without  additional  numbers  I,  II,  III,  etc.,  which  has  led, 
of  course,  to  much  confusion.  Bacterium  Stutzeri  Lehm.  et  Neum.  is  a 
common  and  easily  recognized  denitrifier.  Bad.  fluorescens,  putidum, 
and  radiobacter  are  sometimes  inclined  to  act  in  the  same  manner.  Bac. 
denitrificans  agilis,  first  isolated  by  Ampola  and  Garino  in  Italy,  is,  for 
instance,  a denitrifying  variety  of  Bact.  radiobacter.  Long  continued 
cultivation  in  the  absence  of  nitrate  and  in  the  presence  of  air  usually 
leads  to  the  disappearance  of  this  character,  which  may  be  newly  ac- 
quired, on  the  other  hand,  under  reversed  conditions. 

If  no  other  nitrogen  compounds  are  present  in  the  substrate,  part  of 
the  nitrate  will,  of  course,  be  assimilated.  Under  completely  anaerobic 
conditions  approximately  95  to  98  per  cent  of  the  nitrate  nitrogen  is 
split  off,  and  only  the  remainder  is  assimilated.  The  more  free  oxygen 
will  find  access  to  the  substrate,  the  more  nitrogen  will  be  assimilated, 
and  less  nitrogen  will  escape;  with  full  aeration  the  nitrate  assimilation 
replaces  the  denitrification  entirely. 

The  culture  vessels  shown  in  Fig.  2,  Plate  VII,  may  illustrate  these 
relations.  The  first  flask  to  the  left  contains  a shallow  layer  of  soil 
extract  ammonium-sulfate  -j-  chalk ; accordingly,  nitrification  took 
place.  The  cylinder  next  to  it  contains  a deep  layer  of  soil  extract  + 
nitrate;  no  transformation  took  place,  because  no  suitable  carbon  com- 
pound was  present.  To  the  right  a flask  is  showm  which  contains  a 
shallow  layer  of  soil  extract  -(-  nitrate  -(-  sodium  citrate;  on  account  of 
the  aerobic  conditions  the  nitrate  was  assimilated  by  bacteria  growing  in 
the  solution  and  as  a film  upon  its  surface.  The  same  solution  placed  in 
a deep  layer  in  a cylinder  (to  the  right),  exhibits  very  little  bacterial 
growth,  but  a thick  white  scum  is  formed  by  the  bubbles  of  liberated 
nitrogen. 

The  conditions  under  which  denitrification  takes  place  are  (1) 
Presence  of  nitrate,  (2)  Presence  of  suitable  organic  carbon  compounds, 
(3)  Absence  of  free  oxygen.  Of  course,  sufficient  moisture,  adequate 
temperature,  the  necessary  mineral  salts,  etc.,  must  also  be  available. 
But  tbe  three  conditions  first  mentioned  explain  at  once  why,  with  proper 
management,  the  losses  dim  to  denitrification  in  barnyard  manure  and  in 
soil  can  be  kept  within  fairly  narrow  limits.  A properly  kept  manure 
pile  does  not  offer  a good  substrate  for  nitrification,  which  would  have  to 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  109 


precede  any  denitrification.  And  in  field  soils,  as  a rule,  the  amount  of 
organic  substances  is  so  low  and  the  aeration  so  strong  that  very  little 
denitrification  can  take  place.  Very  wet  soils  and  an  excess  of  organic 
substances  naturally  will  change  the  situation.  Under  such  conditions 
great  losses  may  indeed  be  caused  by  denitrifying  bacteria. 

Nitrogen  Fixation. — The  only  well  known  manner  of  biological 
nitrogen  fixation  is  the  assimilation  of  free  nitrogen  by  certain  bacteria, 
fungi  and  algae.  No  higher  organism  is  able  to  build  up  proteins  from 
elementary  nitrogen,  and  in  view  of  the  losses  in  nitrogen  compounds 
caused  by  the  liberation  of  free  nitrogen,  this  peculiar  capability  of  some 
microorganisms  is  of  fundamental  importance  for  the  continual  restora- 
tion of  the  equilibrium  of  compound  nitrogen  available  for  lower  as  well 
as  for  higher  organisms.  During  the  last  ten  or  twelve  veai's  various 
chemical  methods  have  been  developed  which  make  it  possible  to  pro- 
duce amids  (eyanamid),  ammonia,  or  nitrate  from  the  nitrogen  of  the 
air.  But  the  nitrogen  assimilation  performed  by  bacteria  is  undoubtedly 
of  greater  importance,  because  much  more  nitrogen  is  fixed  in  this 
manner  and  at  a much  lower  cost  than  by  any  technical  process. 

Nitrogen  assimilation  takes  place  only  if  large  quantities  of  easily 
accessible  carbonaceous  substances  are  available.  If  this  source  of  energy 
is  missing,  fixation  of  free  nitrogen  becomes  impossible,  and  the  micro- 
organisms concerned  live  like  all  others  on  various  nitrogen  compounds. 
The  following  types  of  biological  nitrogen  fixation  are  known  at  present : 
(1)  Nitrogen  assimilation  by  the  root  nodule  bacteria  of  leguminous 
plants,  (2)  nitrogen  assimilation  by  microorganisms  living  on  and  in  the 
roots  of  other,  non-leguminous  plants,  (3)  nitrogen  assimilation  by  bac- 
teria living  in  the  leaves  of  certain  tropical  plants,  (4)  nitrogen  assimila- 
tion by  various  bacteria,  fungi,  and  algae  in  the  soil.  In  the  first  three 
cases  higher  and  lower  organisms  live  in  close  symbiosis,  while  there  is  no 
symbiosis  in  the  last  case,  or  only  one  among  microorganisms. 

Nitrogen  Fixation  by  Leguminous  Plants. — As  stated  in  the  dis- 
cussion of  the  history  of  bacteriology  (p.  5),  about  2000  years  ago 
Roman  agricultural  writers  were  fully  aware  of  the  fertilizing  effect 
which  may  be  realized  from  the  cultivation  of  leguminous  plants.  But 
in  the  Far  East  (China  and  Japan)  the  same  knowledge  had  gained 
a foothold  at  still  earlier  times,  as  proved  by  the  use  of  legumes  for  green 
manuring  since  ancient  periods.1  The  same  practice  became  firmly 
established  in  European  agriculture  in  the  course  of  the  nineteenth  cen- 
tury. And  the  more  it  is  applied  to  American  farming  the  more  will 
beneficial  results  be  obtained. 


1 F.  H.  King,  “Farmers  of  Forty  Centuries.' 


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In  1838  the  first  exact  experiments  were  made  by  the  French  chemist 
Boussingault,  which  indicated  that  it  is  the  fixation  of  nitrogen  from 
the  air  which  exerts  the  fertilizing  effect  upon  the  growth  of  leguminous 
plants.1  Since  then  it  has  been  generally  acknowledged  by  European 
farmers  that  the  most  practical  and  economical  means  of  supplying  the 
farm  with  nitrogen  is  by  including  legumes  in  every  crop  rotation.  Most 
scientists,  however,  insisted  on  more  rigorous  tests,  but  these  gave  mostly 
negative  results  during  the  next  decades.  It  was  considered  necessary  to 
heat  the  soil  thoroughly  before  using  it  for  such  experiments,  and  some 
very  careful  investigators  even  covered  the  soil  in  their  pots  with  thick 
layers  of  wax  in  order  to  prevent  absorption  of  ammonia  from  the  air. 
Bacterial  life  was,  of  course,  impossible  under  such  circumstances.  But 
as  early  as  1851  another  French  scientist,  named  Roy,  demonstrated  that 
the  nitrogen  gains  entrance  to  the  legumes  only  through  the  roots,  not 
through  the  leaves,  and  ten  years  later  Bretschneider  discovered 
that  it  was  the  high  temperature  to  which  the  soils  were  exposed,  which 
made  nitrogen  fixation  impossible.  Furthex-more,  in  1858  it  was  noticed 
by  Lachmann  that  the  peculiar  nodules,  characteristic  of  the  roots  of 
leguminous  plants,  are  caused  by  the  invasion  of  motile  bacteria,  which 
remain  therein  as  long  as  the  plant  lives,  and  he  refers  to  the  opinion 
shared  by  many  agriculturists  of  his  time  that  these  nodules  are  the 
organs  of  nitrogen  fixation.  It  is  beyond  doubt  that  a correct  ixxsight 
would  have  been  reached  quickly  if  all  x-esults  obtained  had  been  propei’ly 
considered  and  correlated.  But  the  conclusion  that  nitrogen  fixation 
takes  place  in  the  root  nodules  as  a result  of  bacterial  activity  was  evi- 
dently still  too  new  and  too  strange  to  the  leading  scientists  of  that  time. 

Nevertheless,  xxiore  and  ixxore  positive  findings  were  gathered.  Several 
German  farmers,  who  had  obtained  splendid  results  on  very  poor  sandy 
soils  merely  by  the  cultivation  of  leguminous  plaixts,  stated  once  more 
and  very  firmly  that  nitrogen  fixation  must  be  the  cause  of  this  beneficial 
effect.  That  they  and  the  earlier  investigators  were  perfectly  right  was 
finally  decided  by  thorough  experiments  made  in  the  eighties  of  last 
century  by  Atwater  in  America,  by  Ilellriegel  aixd  Wilfarth  in  Germany, 
and  by  Lawes  and  Gilbert  in  England.  Not  all  authors,  however,  were 
ready  to  be  convinced  by  the  facts  recorded.  The  “new-fangled  bacteria 
hypothesis”  was  still  frequently  ridiculed,  and  even  to-day  it  happens 
from  time  to  time  that  far-fetched  and  quite  unwarranted  theories  are 
put  forward  in  order  to  displace  those  now  firmly  established  results.  T. 
Jamieson  in  Scotland  ascribed,  for  instance,  only  a few  years  ago  the 

1 Complete  references  to  this  historical  summary  are  given  in  Lohxis,  “Handbuch 
der  landwirtschaftlichen  Bakteriologie,”  1910,  pp.  646-650. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  111 


ability  of  nitrogen  assimilation  to  special  hair-like  protrusions  of 
leguminous  and  other  plants,  although  it  had  been  known  since  Roy 
reported  upon  his  experiments  in  1851  that  the  fixation  and  transforma- 
tion of  nitrogen  takes  place  in  the  roots,  not  in  the  aerial  parts  of  the 
legumes. 

Nodule  Bacteria  of  Leguminous  Plants. — The  results  obtained  by 
the  American,  German,  and  British  chemists  mentioned  above,  were  com- 
pleted by  bacteriological  experiments  made  by  M.  W.  Beijerinck  in  Delft 
and  published  in  1888-1891.  He  was  the  first  to  succeed  in  obtaining  pure 
cultures  of  the  nodule  bacteria,  and  was  able  to  demonstrate  tbeir  ability 
to  produce  root  nodules  and  to  assimilate  nitrogen  from  the  air. 
Equally  valuable  investigations  on  the  same  subject  were  made  simul- 
taneously by  A.  Prazmowski  in  Poland;  his  results  were  in  complete 
agreement  with  those  of  the  Dutch  bacteriologist.  But  growth  and 
activity  of  nodule  bacteria  in  pure  culture  are  not  always  satisfactory. 
According  to  their  adaptation  to  symbiotic  life  they  must  find  special  en- 
vironmental conditions,  otherwise  they  will  not  fully  display  their  abili- 
ties. Their  growth  on  the  substrates  ordinarily  used  in  the  laboratories 
is  very  slight,  and  the  same  holds  true  in  regard  to  nitrogen  fixation. 
Several  investigators  could  not  discover  any  nitrogen  assimilation  in 
their  experiments ; others,  however,  secured  positive  results.  Usually 
these  gains  in  nitrogen  are  low  (approximately  2 to  3 mg.  N per  100 
cc.  of  a 1 per  cent  sugar  or  mannite  solution),  but  if  an  arrangement  is 
made  by  which  it  becomes  possible  to  add  at  short  intervals  only  small 
amounts  of  nutrient  solution  and  to  remove  promptly  the  metabolic 
products  from  the  bacterial  growth,  as  is  the  case  in  the  plant,  the  nitro- 
gen fixation  shows  a marked  increase  (from  2 to  3 to  6 to  12  mg.).  Un- 
doubtedly, the  bacterial  activity  within  the  root  nodules  is  still  more 
efficient  and  more  economical,  but  the  results  suffice  to  prove  that  the 
bacteria  themselves  fix  the  nitrogen,  and  that  it  is  erroneous  to  assume, 
as  has  been  done  repeatedly,  that  the  bacteria,  only  “stimulate”  the 
green  plant  in  some  mysterious  manner  so  that  the  latter  acquire  the 
ability  to  assimilate  free  nitrogen. 

Beijerinck  chose  as  scientific  name  of  the  nodule  bacteria  the  desig- 
nation Bacillus  radicicola.  According  to  the  rules  of  scientific  nomencla- 
ture this  species  name  must  be  retained,  although  instead  of  Bacillus 
frequently  the  generic  names  Bacterium  and  Pseudomonas  were  and  are 
used,  since  no  uniform  usage  has  been  established  in  this  respect,  as  was 
discussed  in  Chapter  III.  All  nodule  bacteria  are  temporarily  motile, 
but  the  mode  of  flagellation  was  for  a long  time  a matter  of  dispute. 
Their  gonidia  are  always  monotrichous,  as  was  first  observed  by 
Beijerinck.  But  full  grown  nodule  bacteria  exhibit  two  types  of  flagel- 


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lation.  Those  occurring  in  Trifolium,  Medicago,  Vicia,  Pisum,  and  other 
cultivated  leguminous  plants  of  European  origin  are  peritrichous,  while 
those  living  in  the  roots  of  Soja,  Vigna,  Lespedeza,  and  other  natives  of 
Asia  have  polar  flagella.1  It  is  still  doubtful  whether  changes  from  one 
to  the  other  type  are  possible  or  not;  the  majority  of  observations  made 
in  this  respect  are  against  such  a possibility. 

Two  years  after  Beijerinck  had  published  his  findings,  a German 
botanist,  A.  B.  Frank,  gave  a quite  different  description  of  what  he 
erroneously  believed  to  be  the  nodule  producing  organism,  and  proposed 
the  name  Rhizobium  leguminosarum  for  his  bacterium  which  was 
characterized  by  a yellow  pigment.  It  is,  of  course,  very  incorrect  to  use 
this  name  instead  of  B.  radicicola  Beij.  for  the  genuine  nodule  organism, 
as  was  done  repeatedly. 

From  soil  another  species  was  isolated  by  Beijerinck  which  re- 
sembles B.  radicicola  in  many  respects  rather  closely,  but  which  does 
not  produce  root  nodules.  It  was  named  Bacillus  (or  Bacterium) 
radiobacter.  Its  growth  on  artificial  substrates  is  somewhat  better  than 
that  of  the  nodule  bacteria,  and  because  it  also  often  invades  the  root 
nodules  it  was  repeatedly  isolated  from  there  and  confounded  with  the 
real  nodule  organism.2  Certain  varieties  of  B.  radiobacter  indicate  a rela- 
tionship to  B.  coli  and  to  certain  other  bacteria  common  in  soil  as  well 
as  in  milk.  Branching,  which  was  frequently  noticed  with  nodule  bac- 
teria, can  be  observed  in  all  of  them,  and  it  can  not  be  accepted  as  a 
reason  to  place  B.  radicicola  far  apart  from  those  related  forms,  as  was 
repeatedly  recommended.  In  Chapter  I it  was  emphasized  that  branch- 
ing is  by  no  means  so  rare  among  bacteria  as  was  formerly  thought. 

Nitrogen  Fixation  in  the  Roots  of  Non-leguminous  Plants. — Similar 
nodules  as  are  regularly  found  on  the  roots  of  legumes,  have  also  been 
noted  with  representatives  of  several  other  groups  of  higher  plants.  In 
part  they  are  purely  pathologic,  caused  by  the  intrusion  of  parasitic 
organisms.  But  with  certain  plants  they  are  a constant  and  character- 
istic feature,  just  as  with  the  legumes.  Whether  they  perform  analogous 
functions  is  still  a matter  of  dispute.  Some  positive  indications  were  ob- 
tained, but  very  often  even  the  efforts  to  cultivate  the  causative  organ- 
isms have  failed  entirely.  The  conspicuous  indifference  displayed  by 
these  plants  toward  the  nitrogen  content  of  the  soil  makes  further  ex- 
periments very  desirable.  Some  members  of  this  group  appear  fitted 
for  improving  otherwise  uncultivated  stretches  of  land. 

Such  nodule  bearing  plants,  mostly  trees  and  shrubs,  are  the  alder 

1 F.  Lohnis  and  R.  Hansen,  Jour.  Agric.  Research,  vol.  20,  No.  7,  1921,  p.  543; 
I.  Shunk,  Jour.  Bad.,  vol.  6,  No.  2,  1921,  p.  239. 

2 Lohnis  and  Hansen,  1.  c. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  113 


(Alnus),  several  Elaeagnaceae  (Elaeagnus,  Hippophae,  and  Lepar- 
gyrea),  Podocai’pus,  Myriea  Gale,  Comptonia  peregrina,  Ceanothus, 
Coriaria,  and  Cyeas  species.1  In  the  last-named  case  the  nodules  are 
inhabited  not  only  by  nitrogen  fixing  bacteria  but  also  by  blue  green 
algae  (Nostoc  or  Anabaena).  From  the  other  hosts  several  organisms 
have  been  isolated  which  either  resemble  B.  radicicola  or  exhibit  more  the 
characters  of  an  Actinomyces. 

Various  cruciferous  plants  have  also  been  supposed  to  assimilate 
free  nitrogen,  although  it  is  more  probable  that  they  merely  exhaust 
very  thoroughly  the  supply  of  nitrogen  compounds  present  in  the 
soil.  Nevertheless,  further  investigations  are  needed.  An  Italian 
author  has  stated  that  nitrogen  fixing  bacteria,  related  to  B.  radicicola, 
are  active  in  the  roots  of  such  plants.2 

Many  plants  whose  natural  habitats  are  in  more  or  less  acid  soils  rich 
in  humus,  such  as  the  Ericaceae,  Conifers,  and  others,  are  known  to  have 
various  fungi  growing  on  and  in  their  roots,  forming  a so-called 
mycorrhiza.  Despite  numerous  investigations,  full  light  has  not  yet 
been  shed  upon  the  physiological  value  of  this  symbiosis,  but  it  is  certain 
that  different  functions  are  to  be  considered,  and  it  is  probable  that 
among  them  nitrogen  fixation  plays  its  role.3  Several  Phoma  species  have 
been  found  to  be  capable  of  assimilating  free  nitrogen.4 

Nitrogen  Fixation  in  the  Leaves  of  Tropical  Plants. — Another 
interesting  type  of  symbiosis  between  bacteria  and  higher  plants  was 
more  recently  discovered  in  several  tropical  genera  (Pavetta,  Psychotria, 
Ardisia,  Spathodea,  etc.)  some  of  which  have  been  used  since  ancient 
times,  like  the  legumes,  for  green  manuring.  The  bacteria  again  resem- 
ble in  morphological  and  cultural  characters  B.  radicicola  to  some  extent, 
but  instead  of  producing  nodules  at  the  roots,  they  establish  themselves 
in  the  leaves,  which  are  everywhere  or  only  at  the  edges  covered  by 
small  bead-like  nodules  filled  with  bacteria.  Unlike  B.  radicicola, 
which  does  not  invade  the  stems  and  seeds  of  its  hosts,  this  is  done  by 
those  organisms.  There  is  a great  number  of  them  always  to  be 
found  in  the  seeds,  and  in  view  of  the  difficulty  with  which  the  leaves 
would  otherwise  be  reached  by  the  bacteria,  such  permanent  symbiosis 
and  general  permeation  of  the  host  by  the  bacteria  is,  of  course,  of  con- 

1 K.  F.  Kellerman,  U.  S.  Dept.  Agr.  Yearbook,  1910,  p.  213;  K.  Shibata  and 
M.  Tahara,  Bot.  Mag.,  Tokyo,  vol.  31,  1917,  p.  157. 

2 Cauda,  Nuov.  giorn.  botan.,  vol.  26,  1919,  p.  169. 

3 Peklo,  Zeitschr.f.  Garungsphysiol.,  vol.  2,  1913,  p.  275;  M.  Ch.  Rayner,  Bot.  Gaz., 
vol.  73,  1922,  p.  226;  E.  Melin,  Jour.  Ecol.  vol.  9,  1922,  p.  254. 

4B.  M.  Duggar  and  A.  R.  Davis,  Ann.  Mo.  Bot.  Garden,  vol.  3,  1916,  p.  413; 
Rayner,  1.  c. 


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siderable  advantage.  Nitrogen  fixation  in  pure  cultures  has  been  ob- 
served.1 

Nitrog'en  Fixation  in  the  Soil. — In  addition  to  the  nitrogen  fixation 
caused  by  the  nitrogen  assimilating  microorganisms  living  in  symbiosis 
with  higher  plants,  another  mode  of  nitrogen  fixation,  less  conspicuous 
but  more  general,  takes  place  in  every  soil  due  to  the  activity  of  free 
living  bacteria,  fungi,  and  algae.  As  long  as  thinking  men  have  culti- 
vated the  soil  they  have  been  aware  that  there  is  a general  tendency  in 
all  soils  to  increase  or  to  restore  their  productivity  automatically. 
Barren  rocks,  which  contain  only  minute  traces  of  nitrogen  compounds, 
undergo  a slow  process  of  disintegration  and,  if  the  climate  permits,  they 
transform  themselves  gradually  into  fertile  soils.  Lichens  and  mosses 
appear  first,  herbs  and  shrubs  follow  them,  and  if  the  annual  precipita- 
tion suffices,  ultimately  trees  get  a foothold  and  develop  to  forests,  which 
produce  continuously  enormous  quantities  of  leaves  and  wood  without 
any  fertilization,  provided  that  this  great  natural  productivity  is  not 
destroyed  by  reckless  forest  devastation.  Depressions  in  sterile  sand 
which  hold  some  water  give  rise  to  a growth  of  algae  and  mosses,  which 
in  turn  preserve  more  water  like  a large  sponge,  and  gradually  grow  up 
to  extended  deposits  of  peat.  As  mentioned  above,  nitrogen  fixation  takes 
place  in  certain  herbs,  shrubs,  and  trees  growing  in  the  woods  and  on 
peat  land.  Furthermore,  nitrogen  compounds  are  washed  from  the  air 
into  the  soil  by  rain  and  snow.  But  the  quantities  of  nitrogen  brought 
down  annually  are  not  very  large ; according  to  many  determinations 
made  in  all  parts  of  the  world  an  annual  gain  of  5-10  lbs.  per  acre  may 
be  expected.  This  is  counterbalanced,  however,  by  an  annual  loss  in  the 
drainage  of  about  the  same  magnitude.2 

Many  investigators  have  tried  to  discover  the  causes  of  this  natural 
tendency  of  the  soils  to  restore  or  to  increase  their  fertility.  The  litera- 
ture of  the  last  century  contains  numerous  chemical  hypotheses  which 
have  been  advanced  to  explain  these  gains  in  nitrogen  by  assuming  that 
nitrogen  fixation  take  place  in  soil  under  the  influence  of  humus,  or  of 
iron  and  manganese  oxides,  or  of  ozone  or  of  evaporating  water,  or  of 
weak  electric  currents.  But  exact  experiments  have  shown  that  another 
more  potent  cause  must  be  active,  and  in  1885  it  was  discovered  by  the 
French  chemist  Marcellin  Berthelot  that  this  is  the  nitrogen  assimilation 
performed  by  various  microorganisms  of  the  soil. 

Nitrogen  Assimilating  Organisms  in  the  Soil. — In  1893  the  first  re- 
sults of  investigations  upon  the  nitrogen  fixation  in  pure  cultures  of  soil 

1 F.  C.  von  Faber,  Jahrb.  f.  wissenschaftl.  Bolanik,  vol.  51,  1912,  p.  2S5. 

2Lohnis,  “Handbuch  der  landw.  Bakteriologie,”  1910,  pp.  635,  643. 


Lohnis-Fred,  Text  book 


Plate  VIII 


1.  Growth  of  Azotobacter  on  gypsum  plates,  nat.  size 
a.  Azotobacter  airoococcum  b.  Azotobacter  Beijerinckii 

c.  Azotobacter  vitreum  d.  Azotobacter  agile 


2.  Aerobic  cellulose  decomposition,  nat.  size 
Bacteria  Fungi 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  115 


bacteria  were  published  by  M.  Berthelot,  and  a few  weeks  later  additional 
data  were  furnished  on  the  same  subject  by  the  Russian  bacteriologist  S. 
Winogradsky.  Berthelot  worked  with  different  aerobic  bacteria,  while 
Winogradsky  made  his  experiments  with  an  anaerobic  species  which  he 
called  Clostridium  Pastorianum.  It  was  later  proved  by  Bredemann  that 
this  separate  name  was  not  correctly  applied.  Winogradsky’s  organism 
is  merely  a variety  of  the  common  anaerobic  butyric  acid  bacillus  ( B . 
amylobacter ) which  can  be  isolated  from  every  soil,  and  which  under 
suitable  conditions  always  fixes  some  nitrogen.  The  gains  recorded  by 
Winogradsky  (2  mg.  N per  1 g.  sugar)  were  no  higher  than  those  ob- 
served by  Beijerinck  and  others  in  tests  of  B.  radicicola.  Later  experi- 
ments showed  gains  up  to  6 mg.  per  1 g.  sugar. 

Still  greater  activity  is  usually  displayed  by  Azotobacter,  the  large 
aerobic  nitrogen  fixing  organism  described  by  Beijerinck  in  1901,  which 
is  shown  in  Fig.  5,  Plate  I.  10  to  15  mg.  N per  1 g.  sugar  are 
frequently  assimilated  in  routine  experiments  with  these  bacteria,  but 
much  larger  gains  can  be  obtained  under  more  favorable  conditions, 
especially  if  very  young  and  vigorous  cultures  are  tested.1  Dextrose  and 
mannitol  are  generally  the  best  kinds  of  carbonaceous  food,  but  numerous 
other  carbohydrates,  alcohols,  and  organic  salts  can  be  used.  The  prod- 
ucts of  the  decomposition  of  cellulose  and  of  pectic  substances  are 
equally  accessible  to  Azotobacter ; the  symbiosis  of  such  groups  of  organ- 
isms is  undoubtedly  of  great  benefit  for  the  nitrogen  fixation  in  soil. 
Various  species  or  varieties  of  Azotobacter  have  been  isolated;  four  of 
them  are  shown  on  Plate  VIII.  Beijerinck  described  two  of  them: 
Azotobacter  chroococcxim,  characterized  by  its  brown  or  black  color,  and 
Azotobacter  agile,  a rapidly  motile  form,  producing  in  agar  as  well  as 
in  solution  a brilliant  green  fluorescence.  A.  Vinelandii,  isolated  by  J. 
G.  Lipman,  is  identical  with  A.  agile.  A.  Beijerinckii  is  a variety  of  A. 
chroococcum,  usually  growing  white  but  in  certain  stages  of  its  develop- 
ment distinctly  yellow.  A.  vitreum  has  never  shown  either  pigmentation 
or  motility,  yet  it  seems  to  be  a variety  of  A.  agile.  The  pleomor- 
phism  of  this  group  of  organisms  is  very  conspicuous ; laboratory  cul- 
tures often  change  from  the  large  cell  form  to  small  coccoid  and  rod 
shaped  types.2 

During  the  last  twenty  years  numerous  other  aerobic  bacteria  have 
been  isolated  from  soils,  which  proved  to  be  able  to  assimilate  free 
nitrogen.  Various  cocci,  non-sporulating  and  sporulating  rods  have  been 
described.  Several  of  them  were  recently  found  to  be  growth  types  of 

1 A.  Koch  und  S.  Seydel,  Ceniralbl.  f.  Bald.,  II.  Abt.,  vol.  31,  1911,  p.  570. 

2F.  Lohnis  and  N.  R.  Smith,  Jour.  Agric.  Research,  vol.  6,  1916,  p.  675;  vol.  23, 
1923,  No.  6. 


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Azotobacter,  therefore  they  are  especially  numerous  in  soils  where 
the  large  cells  of  Azotobacter  are  temporarily  scarce  or  absent.  Bad. 
ladis  viscosum , which  causes  ropiness  in  milk,  is  one  of  them ; the  sporu- 
lating  B.  petasites  is  another  such  form.  Bad.  radiobader , which  was 
mentioned  above,  is  equally  able  to  assimilate  free  nitrogen. 

Green  algae  (Chlorophyceae)  and  blue  green  algae  (Cyanophyceae) 
have  also  been  tested  repeatedly  in  regard  to  their  abilities  to  fix  nitrogen, 
in  most  cases  with  negative  results.  Nevertheless,  they  seem  to  partici- 
pate actively  in  this  process  under  natural  conditions  and  further  experi- 
ments may  prove  more  enlightening.  Small  amounts  of  nitrates  are 
useful  in  starting  algal  growth,  and  later  the  algae  are  able  to  assimi- 
late free  nitrogen.1 

Nitrogen  fixation  in  distinctly  acid  soils  of  high  humus  content  seems 
to  be  due  mainly  to  the  presence  of  nitrogen  assimilating  fungi,  but  again 
most  experiments  thus  far  made  with  pure  cultures  have  failed  to 
furnish  a satisfactory  explanation.  However,  negative  results  should 
not  be  overrated,  as  is  sometimes  done.  Experiments  made  with  Azoto- 
bacter or  Amylobacter  do  also  not  always  show  gains  in  nitrogen. 

Importance  of  Nitrogen  Fixation. — Despite  the  various  doubtful 
points  which  are  awaiting  elucidation  by  future  research,  enough  is 
known  at  present  to  secure  much  insight  into  the  role  played  by  nitrogen 
assimilating  organisms  in  the  soil.  Because  of  the  great  number  of 
species  which  are  able  to  act  in  this  manner,  if  conditions  are  favorable, 
it  is  evident  that  some  nUrogen  assimilation  may  be  expected  in  every 
soil,  as  was  indicated  by  practical  experience.  But  the  relation  between 
carbon  used  and  nitrogen  fixed  is  always  very  wide ; usually  less,  rarely 
more  than  1 part  of  nitrogen  is  assimilated,  while  100  parts  of  car- 
bonaceous material  are  oxidized.  Most  soils,  however,  are  not  very  rich 
in  organic  substances,  and  as  far  as  these  are  available  many  micro- 
organisms, which  do  not  fix  nitrogen,  take  their  share,  too.  It  is  obvious 
that  Azotobacter,  Amylobacter,  and  their  kind  can  never  gather  such 
large  quantities  of  nitrogen  in  the  soil,  as  can  B.  radicicola  in  the  roots 
of  the  legumes.  A steady  stream  of  soluble  carbohydrates  is  here  fur- 
nished by  the  host  plant  almost  exclusively  for  the  use  of  its  symbiont, 
and  the  continual  removal  of  the  products  of  nitrogen  assimilation  by  the 
plant  tends  to  keep  the  efficiency  of  the  bacteria  at  its  maximum.  Nu- 
merous tests  have  shown  that  100  to  200  lbs.  of  nitrogen  can  be  gathered  in 
a good  crop  of  leguminous  plants  per  acre,  while  in  the  soil  itself  onlv 
Vs  or  V10  of  these  quantities  can  be  assimilated,  even  if  the  conditions 
pre  very  favorable.  Occasionally  the  meager  supply  of  organic  sub- 


1 F.  B.  Wann,  Science  N.  S.,  vol.  51,  1920,  p.  247. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  117 


stances  furnished  by  the  soil  may  be  supplemented  to  some  extent  by 
carbohydrates  produced  by  soil  algae,  winch  may  enter  into  symbiotic 
relations  with  Azotobacter  and  other  nitrogen  assimilating  bacteria.  Such 
cooperation  has  repeatedly  been  observed  in  water,  as  well  as  in  semi-arid 
soils;  but  also  in  such  cases  the  actual  gains  can  not  be  very  large.  A 
glance  at  a field  of  alfalfa,  soy  beans,  or  clover  demonstrates  at  once  that 
very  great  quantities  of  organic  substances  must  be  available  in  order  to 
secure  a comparatively  large  amount  of  nitrogen.  Additional  data  will 
be  given  in  Chapter  XIY,  2. 

4.  THE  CYCLE  OF  CARBON,  OXYGEN,  AND  HYDROGEN 

The  assimilation  of  carbon  dioxide  by  green  plants  is  the  fundamental 
reaction  that  leads  to  the  formation  of  all  carbonaceous  substances  which 


participate  in  the  construction  of  plants  and  animals.  A few  bacteria 
are  able  to  perform  a similar  synthetic  process,  but  the  vast  majority  of 
microorganisms  acts  exclusively  in  the  reverse  direction.  Organic  com- 
pounds are  oxidized  by  bacteria  and  fungi  to  carbon  dioxide  and  water 
in  a manner  analogous  to  the  respiratory  process  of  the  higher  organisms. 
However,  not  all  microorganisms  live  in  the  presence  of  air,  and  it  is 
self-evident  that  under  anaerobic  conditions  other  reactions  take  place, 
leading  to  various  intermediate  compounds.  But  sooner  or  later  these  too 
will  be  mineralized  by  oxidizing  microorganisms,  so  that  the  cycle  of 
constructive  and  destructive  processes  will  be  completed. 

Cycle  of  Carbon,  Oxygen,  and  Hydrogen. — Figure  30  illustrates  the 
different  possibilities  known  at  present.  Besides  the  reactions  taking 
place  between  the  organic  carbon  compounds,  carbon  dioxide,  and  water, 
there  are  others  by  which  several  gases  (carbon  monoxide,  methane,  and 
hydrogen)  or  certain  solid  substances  (humus  and  coal)  are  produced 


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as  results  of  incomplete  oxidation,  and  therefore  subject  to  further  trans- 
formation to  carbon  dioxide  and  water. 

In  regard  to  the  organic  carbon  compounds  it  is  the  metabolism  and 
final  oxidation  of  carbohydrates,  fats,  alcohols,  and  organic  acids  which 
are  of  greatest  practical  importance.  Benzol  derivatives  (phenols,  etc.) 
may  also  be  attacked  by  microorganisms,  but  these  substances  are  gen- 
erally much  more  resistant,  and  play  a much  less  conspicuous  role  than 
the  aliphatic  compounds,  as  far  as  quantities  are  concerned.  Because 
carbohydrates  take  part  in  the  formation  of  organic  nitrogenous  com- 
pounds, they  may,  of  course,  appear  as  by-products  in  the  course  of 
bacterial  destruction  of  proteins,  or  they  may  give  rise  to  carbon  dioxide 
and  water. 

What  is  called  humus  by  the  agriculturist  is  a complex  and  highly 
variable  mixture  of  many  organic  as  well  as  inorganic  components.  The 
chemical  reactions  taking  place  in  the  formation  and  destruction  of 
humus  in  the  soil  are  therefore  not  so  well  defined  as  they  are  with  other 
organic  substances.  But  because  of  the  very  prominent  role  they  play 
in  all  soils,  careful  consideration  will  have  to  be  given  to  them,  too.  As 
is  indicated  in  Fig.  30,  part  of  the  humus  may  be  used,  especially  by 
fungi,  to  produce  well-defined  organic  substances  within  their  own  cells; 
the  rest  is  slowly  oxidized  to  carbon  dioxide  and  water,  or  it  is  further 
reduced  to  coal-like  material,  such  as  is  to  be  found  in  deep  peat  deposits 
of  recent  or  ancient  date.  A gradual  loss  of  oxygen  and  hydrogen  to- 
gether with  a continual  enrichment  in  carbon  characterizes  this  process, 
which  is,  of  course,  not  dependent  on  bacterial  life.  It  has  been  repeat- 
edly ascertained,  however,  that  in  the  slow  oxidation  which  is  noticeable 
in  stored  coal  bacteria  may  participate.  But  this  process  is  of  very  little 
practical  importance  compared  with  the  quick  and  nearly  complete  oxi- 
dation taking  place  in  the  burning  of  coal. 

On  the  other  hand,  much  of  the  carbon  monoxide,  methane,  and  hydro- 
gen occurring  in  nature  is  not  produced  by  bacteria,  but  is  the  result  of 
volcanic  activity  and  of  incomplete  combustion  of  coal.  Numerous  bac- 
teria, however,  participate  in  this  process,  and  others,  besides  spontaneous 
oxidation,  complete  the  transformations,  making  use  of  those  gases  for 
respiration  as  well  as  for  assimilation. 

Transformation  of  Sugar  and  of  Starch. — The  carbohydrates 
formed  in  green  plants  are  used  as  far  as  possible  for  human  and  animal 
nutrition.  This  holds  true  especially  in  regard  to  sugar  and  starch, 
which,  if  they  can  not  be  used  immediately,  must  always  be  protected 
against  the  attacks  of  microorganisms  by  heating,  drying,  or  otherwise. 
In  certain  cases,  however,  a partial  transformation  of  sugar  and  starch 
by  bacterial  activities  proves  useful  and  is  therefore  desired,  for  in- 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  119 


stance  in  the  ripening  of  cream  and  of  cheese,  in  the  preparation  of 
silage,  sauerkraut,  etc.  It  is  the  anaerobic  transformation  of  sugar  into 
lactic  acid  which  is  of  greatest  importance  in  these  cases ; the  acid  pro- 
duced acts  as  a preservative  and  at  the  same  time  increases  the  palata- 
bility  of  the  food.  In  such  cases  varying  quantities  of  other  organic 
acids  are  also  regularly  formed.  Formic,  acetic,  propionic,  butyric,  and 
succinic  acids  are  most  common.  Acetic  and  butyric  acids  are  some- 
times very  noticeable  in  spoiled  silage,  where  either  aerobic  acetic  and 
butyric  acid  bacteria  may  act,  if  the  material  is  not  properly  packed  and 
protected  from  the  air,  or  anaerobic  organisms  may  cause  such  deteriora- 
tion, as  they  also  do  sometimes  in  faulty  cheese.  Before  starch  is  acidi- 
fied it  is  hydrolyzed  to  sugar  by  the  amylolytic  (or  diastatic)  action  of 
various  microorganisms.  Many  sporulating  bacilli,  as  well  as  actinomy- 
cetes  and  molds,  are  active  in  this  direction. 

Lactic  Acid  Bacteria. — Several  hundreds  of  so-called  species  of 
lactic  acid  bacteria  have  been  described,  capable  of  transforming  the 
various  sugars  into  either  dextro,  or  laevo-lactic  acid,  or  into  the  inactive 
modification.  Smaller  or  larger  quantities  of  by-products,  mostly  acetic 
and  succinic  acids,  are  formed,  but  this  function  can  not  be  used  for 
classifying  the  lactic  acid  bacteria,  because  it  is  too  unstable  and  always 
influenced  by  the  changing  environmental  conditions.  All  lactic  acid  bac- 
teria of  practical  importance  can  be  divided  into  the  following  four 
groups : 

(1)  Lactic  acid  streptococci  ( Streptococcus  lactis ); 

(2)  Lactobacilli  ( Bacterium  casei); 

(3)  Intestinal  lactic  acid  bacteria  {Bad.  coli,  B.  aerogenes,  and  B.  acidi  lactici) ; 

(4)  Lactic  acid  micrococci  {Micrococcus  lactis  acidi). 

Certain  sporulating  bacilli  and  vibrios,  for  instance  the  cholera  vibrio, 
are  also  able  to  produce  some  lactic  acid,  but  they  are  of  no  interest  in 
this  connection.  The  lactic  acid  streptococci  and  lactobacilli  stand  first 
in  importance,  and  they  were  therefore  separated  by  some  authors  as  the 
“true”  lactic  acid  bacteria  from  the  two  other  groups,  which  were 
classed  as  “pseudo ’’-lactic  acid  bacteria.  It  is  to  be  admitted  that  in 
regard  to  quantity,  as  well  as  to  quality,  generally  the  production  of 
lactic  acid  is  much  more  conspicuous  in  the  two  groups  first  mentioned. 
But  representatives  of  the  two  other  groups  are  also  very  active  acid 
producers  in  the  intestinal  tract,  as  well  as  in  milk  and  dairy  produce. 
One  type  belonging  to  the  third  group,  described  by  Hueppe  as  B.  acidi 
lactici,  was  for  a long  time  considered  to  be  the  most  important  lactic 
acid  organism,  and  lactic  acid  micrococci  are  by  no  means  rare  in  butter 
as  well  as  in  cheese.  Because  of  their  inclination  to  live  within  the  diges- 


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tive  tract  many  intestinal  lactic  acid  bacteria  display  characters  (strong 
gas  formation  and  production  of  disagreeable  flavors)  which  are  detri- 
mental to  milk,  butter  and  cheese.  The  lactic  acid  micrococci,  on  the 
other  hand,  are  closely  related  to  other  non-acid  producing  cocci  which 
possess  strong  proteolytic  abilities.  Frequently  both  functions,  acid  pro- 
duction and  proteolysis,  may  be  exerted  by  the  same  strain  of  micrococci 
according  to  circumstances. 

The  lactic  acid  streptococci  do  not  always  grow  as  typically  globular 
cells,  and  for  a long  time  they  have  been  classed  as  rods.  The  famed 
British  surgeon  John  Lister  published  the  first  accurate  description  of 
such  an  organism  in  1878  and  named  it  Bacterium  lactis.  Some  of  his 


BACTERIUM  LACTIS. 

In  curdled  Milk  \ 

8/ 

after  3 days.  f *'* 

4 

In  unboiled  Urine  o 

0 

• 

0 

0 

after  2 days.  Q { 

0 % 
0 

: 

In  Milk  diluted 

?\  * 

with  1200  parts  of  Water. 

Vy 

S 

after  3 days. 

O'/ 

U** 

? 1 * * T * JrvilA,  in.  Tejis-t/ioitsajutths  of  an  Inch. 

Fig.  31. — Bacterium  lactis  Lister,  from  “Transactions  of  the  Pathological  Society  of 
London,”  Vol.  XXIX,  1878,  Plate  XX. 

drawings  are  reproduced  in  Fig.  31.  The  variability  of  the  cell  form  is 
clearly  demonstrated;  other  illustrations  are  given  as  Figs.  2 and  3 on 
Plate  I.  About  twenty  years  later  the  same  organism  was  described  as 
Bacterium  lactis  acidi  by  Leichmann,  and  this  name  has  been  widely 
used  in  the  dairy  literature  until  quite  recently.  Many  other  names  have 
been  proposed,  usually  on  account  of  rather  unimportant  differences  of 
the  strains  studied.  At  present,  however,  it  is  almost  generally  admitted 
that  this  group  of  organisms  should  be  classed  as  streptococci,  and  that 
the  type  species  should  bear  the  name  Streptococcus  lactis. 

The  lactobacilli  grow  mostly  as  slender  rods  of  considerable  length 
(Fig.  7,  Plate  I)  and  are  much  inclined  to  produce  globular  regenerative 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  121 


bodies  (Fig.  1,  Plate  II).  While  lactic  acid  streptococci  predominate  in 
sour  milk,  cream,  butter,  and  in  soft  cheese,  lactobacilli  are  usually  more 
prevalent  in  silage  and  in  hard  cheese.  Unfortunately,  Leiehmann  named 
one  of  these  forms  Bacillus  lactis  acidi,  as  a counterpart  to  his  Bacterium 
lactis  acidi  mentioned  above.  But  because  the  names  Bacillus  and  Bac- 
terium are  frequently  used  rather  indiscriminately,  much  confusion  has 
arisen  and  persisted  for  a long  time  in  the  literature.  It  seems  best  to 
use  the  name  Bacterium  or  Lactobacillus  casei  as  that  of  the  typical  rep- 
resentative of  this  group. 

Naturally  every  strain  of  the  lactic  acid  bacteria  displays  some  fea- 
tures which  frequently  have  been  considered  sufficient  reason  to  create 
additional  “new  species,”  but  a careful  comparison  of  all  these  descrip- 
tions leaves  no  doubt  that  only  the  four  groups  mentioned  are  fairly  well 
defined.  Each  of  them  can  be  subdivided,  according  to  milk  coagulation, 
gas  formation,  slime  production,  etc.,  into  several  types,  wherein  the 
several  hundreds  of  so-called  species  of  lactic  acid  bacteria  find  their 
places  as  more  or  less  closely  related  varieties.1 

It  is  noteworthy  in  this  connection  that  the  lactic  acid  streptococci  as 
well  as  the  micrococci  are  closely  related  to  pathogenic  species,  namely 
Streptococcus  pyogenes  and  Micrococcus  pyogenes,  and  that  the  same 
holds  true  in  regard  to  the  intestinal  lactic  acid  bacteria ; B.  coli  being 
related  to  B.  typhosus,  and  B.  aerogenes  to  B.  pneumoniae.  These  facts 
are  of  great  importance  especially  as  far  as  the  microflora  of  the  udder 
is  concerned  (see  Chapter  IX,  1). 

Streptococci  and  lactobacilli  generally  grow  best  under  anaerobic  con- 
ditions, while  the  micrococci  are  distinctly  aerobic.  Certain  varieties 
of  the  lactic  acid  bacteria  act  only  upon  sucrose  (in  silage),  others 
only  on  lactose  (in  dairy  produce),  while  many  strains  acidify  these  two 
sugars  as  well  as  others.  But  these  behaviors  are  also  subject  to  much 
variation. 

Formation  of  Slime  from  Carbohydrates. — Milk,  cream,  bread, 
potatoes,  and  other  substances  rich  in  sugar  or  in  starch  become  occa- 
sionally more  or  less  slimy  or  ropy.  The  carbohydrates  contained  therein 
are  consumed  by  microorganisms  which  make  use  of  them  for  construct- 
ing large  slime  capsules  around  their  cells,  such  as  are  shown  in  Fig.  10, 
Plate  II.  This  peculiar  behavior  is  not  rare  among  the  members  of  all 
four  groups  of  lactic  acid  bacteria ; especially  the  Streptococcus  cultures 
used  as  starters  for  cream  ripening  display  sometimes  a marked 

1 Lohnis,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  18,  1907,  p.  97-149;  “Handbuch  der 
landw.  Bakteriologie,”  1910,  p.  192-202;  S.  Orla-Jensen,  “The  Lactic  Acid  Bac- 
teria,” Mem.  Acad.  Copenhagen,  Natur.  and  Mathem.  Sci.  Cl.,  8th  Ser.,  vol.  5,  No.  2, 

1919. 


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tendency  to  degenerate  in  this  manner.  Oidium  lactis  is  also  known  to 
give  rise  occasionally  to  slime  producing  varieties. 

Slimy,  slightly  acid  milk  or  cream  are  well  liked  in  certain  localities, 
tor  instance  the  so-called  “taette”  or  “taettemjolk”  (literally  “tight 
milk”)  in  Norway  and  Sweden,  the  “long  whey”  (that  is,  stringy 
whey)  in  Holland,  and  the  “fiili”  or  “puma”  prepared  by  Finnish 
settlers  in  Minnesota.1  Usually  however  this  alteration  of  milk  and 
cream  is  distinctly  disliked,  although  the  food  value  is  not  much  im- 
paired. The  slime  producing  varieties  of  lactic  acid  bacteria  have  received 
several  specific  names,  of  which  the  following  ones  are  frequently  used 
for  members  of  the  first,  third  and  fourth  group  of  the  lactic  acid  bacteria, 
respectively:  Streptococcus  hollandicus,  Bacterium  lactis  viscosum,  and 
Micrococcus  pituitoparus.  The  so-called  Leuconostoc  (Fig.  10,  Plate  II) 
which  has  played  a rather  disadvantageous  role  in  sugar  factories,  is 
another  slime  producing  Streptococcus  variety.  Its  action  can  be  sup- 
pressed easily  by  keeping  the  temperature  of  the  liquids  permanently  at 
or  above  60°  C.  In  the  dairy  a thorough  disinfection  of  all  utensils  is 
usually  sufficient  to  eliminate  the  trouble.  Sometimes,  however,  the  water 
contains  slime  producing  organisms,  mostly  Bad.  lactis  viscosum  which 
is  common  in  soil.  Such  water  must  be  boiled,  if  no  supply  of  pure 
water  is  available. 

In  starchy  food,  especially  in  bread,  slime  production  is  always  due  to 
the  activity  of  certain  sporulating  bacilli  ( B . mesentericus ) which  may 
also  become  detrimental  in  sugar  factories.  Because  of  their  great  re- 
sistance, part  of  the  spores  survive  the  high  temperatures  in  the  baking 
oven,  and  they  may  afterwards  cause  a very  disagreeable  spoilage  of  such 
bread,  if  this  is  not  kept  at  a low  temperature.  Addition  of  acid  (sour 
milk  or  leavens)  proves  helpful,  too,  because  the  spores  do  not  germinate 
in  an  acid  substrate. 

Formation  of  Alcohol. — The  acidification  of  carbohydrates  is  always 
accompanied  in  nature  by  the  formation  of  alcohol.  Almost  invariably  a 
symbiosis  exists  between  lactic  acid  bacteria,  mostly  lactobaeilli,  and 
ethyl-alcohol  producing  yeasts.  Furthermore,  several  bacteria  are  able 
to  produce  acids  as  well  as  ethyl  and  other  alcohols.  Mannitol,  for  in- 
stance, is  a by-product  of  the  metabolism  of  many  lactic  acid  bacteria. 
The  quantities  of  alcohol  may  be  small,  as  in  sour  milk,  cream,  and 
cheese,  or  moderate,  as  in  sauerkraut  and  in  silage,  or  large,  as  in  the 
mash  used  in  the  fermentation  industries.  The  various  alcohols  con- 
tribute, in  combination  with  different  acids,  very  materially  to  the  flavors 
of  dairy  products  and  of  silage.  Certain  oriental  tribes  prepare  peculiar 

1 H.  Macy,  Abstracts  of  Bacteriology,  vol.  6,  1922,  p.  18. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  123 


fermented  milks,  such  as  kefir  and  koumiss,  to  which  even  curative 
qualities  are  ascribed,  and  which  therefore  are  used  to  some  extent  in 
European  as  well  as  in  American  sanatoriums.  Koumiss  can  be  properly 
prepared  only  from  mares’  milk,  which  gives  a specially  flavored  and 
much  more  alcoholic  drink  than  can  be  obtained  from  cows’  milk.  Still 
stronger  drinks  are  produced  in  Siberia  by  distilling  the  koumiss.1 

Transformation  of  Fats. — Fats  in  milk,  cream,  butter,  and  in  cheese, 
as  well  as  in  concentrated  feeding  stuffs  (rape  cakes,  etc.)  may  be  more 
or  less  thoroughly  transformed  and  destroyed  by  bacteria  as  well  as  by 
molds.  As  fats  are  compounds  of  organic  acids  (butyric,  oleic,  stearic, 
and  other  acids)  with  glycerol,  an  alcohol  of  high  nutritive  value,  it  is 
easily  understood  why  many  bacteria  are  inclined  to  split  these  sub- 
stances, making  use  of  the  glycerol  as  a source  of  carbon,  but  leaving 
the  free  fatty  acids  which  make  those  products  more  or  less  rancid. 
Oxidative  processes  may  participate  in  the  deterioration  of  the  flavor  of 
butter  and  cheese ; sunlight  and  air  themselves  are  able  to  exert  such  detri- 
mental effects  upon  butter  fat.  As  far  as  organisms  are  concerned  they 
are  almost  exclusively  aerobic  bacteria  and  fungi.  The  latter  do  not  make 
use  of  the  glycerol  only,  but  they  destroy  the  fatty  acids  too,  thereby 
producing  considerable  quantities  of  carbon  dioxide  and  water,  as  was 
illustrated  by  the  figures  given  on  p.  45. 

Butyric  Acid  Bacilli. — The  peculiar  flavor  of  rancid  foodstuffs  is 
due  mostly  to  the  presence  of  free  butyric  acid.  This  may  be  derived 
from  fat;  but  there  are  numerous  aerobic  as  well  as  anaerobic  bacteria 
capable  of  producing  butyric  acid  from  carbohydrates.  Diseased  pota- 
toes stored  in  pits  are  often  destroyed  by  a rapidly  progressing  butyric 
acid  fermentation  which  transforms  their  substance  into  an  evil  smelling 
viscous  liquid.  The  most  active  butyric  acid  bacteria  are  anaerobic.  As 
usual,  numerous  species  have  been  created,  but  there  is  little  doubt  that 
most  of  them  are  in  fact  merely  varieties  of  one  species  which  has  re- 
ceived several  names;  Bacillus  amylobacter  or  Clostridium  butyricum 
are  the  designations  most  commonly  used.  These  bacilli  produce  rather 
large  endospores  which  frequently,  though  not  always,  cause  a swelling 
of  the  sporulating  cell,  so  that  it  presents  a club-like  appearance  (Fig.  12, 
Plate  II).  It  is  because  of  this  peculiarity  that  the  genus  name 
Clostridium  was  introduced,  but  as  this  feature  is  by  no  means  con- 
stant the  name  is  not  well  founded.  Variations  are  frequent  within  this 
group  in  two  directions.2  Either  spore  formation  as  well  as  motility 
vanish  and  the  production  of  butyric  acid  is  largely  replaced  by  the  for- 

1B.  Rubinsky,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  28,  1910,  p.  161-219. 

2 Grassberger  und  Schattenfroh,  Archiv  f.  Hyg.,  vol.  60,  1907,  pp.  40-78;  G. 
Bredemann,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  23,  1909,  pp  385-568. 


124 


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mation  of  other  acids,  mostly  lactic  acid,  or  the  spores  appear  in  ter- 
minal position  (so-called  Plectridium  type)  and  instead  of  acidification 
a proteolytic  action  becomes  more  and  more  noticeable.  In  the  first  case 
the  relation  to  the  lactobacilli  becomes  evident,  while  in  the  second  ease 
the  general  character  is  more  or  less  similar  to  that  of  the  anaerobic 
putrefying  bacteria,  B.  putrificus  and  related  forms.  All  these  relations 
between  the  various  groups  of  anaerobic  bacteria  can  be  illustrated  in  the 
following  manner: 


B.  putrificus  B.  amylobacter 

and  related  forms  and  related  forms 


Immotile,  non-sporulating 
butyric  acid  bacteria 

I 

Lactobacilli 

It  is  self-evident  that  profound  changes  in  the  general  character 
can  be  observed  only  in  experiments  of  long  duration.  Short  termed  in- 
vestigations may  easily  lead  to  the  erroneous  conclusion  that  one  or  the 
other  character  is  quite  constant.  On  such  basis,  scores  of  species  and 
dozens  of  genera  of  anaerobes  have  been  proposed  which  are  not  tenable. 

The  aerobic  butyric  acid  bacilli  are  related  to  the  Amylobacter  group 
as  well  as  to  the  common  hay  and  potato  bacilli,  B.  subtilis  and  B.  mesen- 
tericus.  They  play,  as  a rule,  an  inconspicuous  role  in  nature. 

Decomposition  of  Pectic  Substances. — Wherever  plant  residues  are 
decomposed  in  nature  the  dissolution  of  their  pectic  substances  represents 
an  important  step,  because  these  compounds  participate  largely  in  uniting 
the  separate  cells  into  solid  tissues.  If  they  are  dissolved  a general  dis- 
integration takes  place,  as  is  noticeable,  for  instance,  in  potatoes  under- 
going the  butyric  acid  fermentation. 

A particular  type  of  disintegration  takes  place  in  the  retting  of  flax, 
hemp,  and  other  similar  plants  used  in  the  textile  industry.  It  is  de- 
sired in  these  cases  that  in  addition  to  sugar  and  starch  the  pectic  sub- 
stances be  dissolved,  but  that  the  cellulose  which  forms  the  fibers,  shall 
remain  untouched.  Chemical  as  well  as  biological  methods  are  available 
for  this  purpose.  The  latter  make  use  of  either  anaerobic  or  aerobic  or- 
ganisms, which  in  most  cases  are  again  closely  related  to  the  butyric  acid 
bacteria  just  discussed.  The  forms  active  under  anaerobic  conditions 
are  usually  called  Plectridium  or  Granulobacter  pectinovorum ; but  it 
has  been  proved  that  they  are  varieties  of  B.  amylobacter.1  If 
the  retting  takes  place  in  the  presence  of  air  certain  sporulating  bacilli, 

1 G.  Bredemann,  Centralbl.f.  Bakt.,  II.  Abt.,  vol.  23,  1909,  p.  3S5-56S. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  125 


related  to  B.  subtilis  and  B.  mesentericus,  may  become  active,  or  various 
molds  may  enter  into  the  process;  but  these  fungi  are  very  likely  to 
destroy  simultaneously  part  of  the  cellulose  and  cause  thereby  a deterio- 
ration in  the  quality  of  the  fibers. 

Generally  the  most  satisfactory  results  are  obtained  if  the  retting 
proceeds  anaerobically  under  water  and  at  a somewhat  elevated  tem- 
perature. Besides  gases  the  bacteria  produce  various  organic  acids, 
which  must  be  either  neutralized  or  completely  removed.  In  the  latter 
case  the  water  is  constantly  changed;  in  the  former  neutralizing  sub- 
stances, such  as  lime,  soda,  or  potash,  are  added  to  the  water. 

Cellulose  Decomposition. — The  final  step  in  the  decomposition  of 
plant  tissues  is  represented  by  the  destruction  of  the  cellulose,  generally 
the  most  resistant  part  of  the  vegetable  cells.  Again  the  process  may 
occur  under  anaerobic  or  under  aerobic  conditions ; usually  it  is  more 
rapid  in  the  latter  case. 

The  organisms  responsible  for  the  anaerobic  cellulose  decomposition 
are  not  yet  fully  known.  The  problem  has  been  studied  by  V.  Omelianski 
of  Petrograd,  Russia,  who  described  two  types  of  slender  bacilli  with 
terminal  spores,  which,  however,  could  not  be  grown  in  pure  culture. 
One  was  characterized  by  its  ability  to  produce  methane  in  addition  to 
carbon  dioxide  and  various  fatty  acids,  while  the  other  evolved  hydrogen.1 
Examinations  of  the  original  cultures,  made  by  K.  F.  Kellerman  and  his 
collaborators,2  did  not  confirm  the  findings  of  the  Russian  bacteriologist. 
Only  aerobic  cellulose  bacteria  could  be  found  in  Omelianski ’s  cultures 
as  well  as  in  soil  and  manure.  Therefore,  new  investigations  will  have 
to  be  made  in  order  to  explain  these  differences. 

The  first  step  in  the  decomposition  of  cellulose  is  its  transformation 
into  cellobiose  and  glucose.  These  sugars  are  quickly  attacked  by  nu- 
merous bacteria  which  produce  acids  and  gases,  and  it  is  very  essential 
that  these  by-products  be  continually  removed  if  a rapid  cellulose  de- 
composition under  anaerobic  conditions  is  to  take  place.  Figure  32  dem- 
onstrates the  different  results  obtainable  in  such  experiments.  In  the 
intestines  as  well  as  in  the  manure  pile  the  intermediate  products  are 
quickly  destroyed  by  other  microorganisms,  provided  that  the  reaction  is 
alkaline.  The  bacterial  processes  are  in  both  cases  favored  by  relatively 
high  temperatures.  Even  at  50°  C.  the  decomposition  of  cellulose  is 
fairly  rapid,  due  to  the  participation  of  thermophilic  organisms. 

If  the  decomposition  proceeds  in  the  presence  of  air,  as  in  soil,  vari- 
ous non-sporulating  as  well  as  sporulating  bacteria  and  also  different 

1 Omelianski,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  8,  1902,  p.  324;  vol.  11,  1904,  p.  369. 

2 Kellerman  and  McBeth,  Centralbl.  f.  Bakt.,  II.  Abt.,  34,  1912,  p.  485;  Same, 
Scales  and  N.  R.  Smith,  1.  c.,  vol.  39,  1913,  p.  502. 


126 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


fungi  may  become  active.  Figure  2,  Plate  VIII,  shows  the  growth  of 
such  bacteria  and  fungi  on  paper.  The  fungi  produce  dark  humus- 
like substances,  whereas  the  bacteria  dissolve  the  cellulose,  as  a rul°, 
completely,  and  oxidize  at  the  same  time  the  intermediate  products  to 
carbon  dioxide  and  water.  If  nitrate  is  present,  denitrifying  bacteria 


Fig.  32. — Decomposition  of  cellulose  in  solution  (left)  repeatedly  changed,  or  (right) 

not  changed. 

may  act,  making  use  of  the  oxygen  of  the  nitrate  for  the  oxidation  of 
the  cellulose,  according  to  the  formula: 

(C6Hio05)x+8  KNO2  =4  KHCO3  + 2 K2CO3+4  N2+3  H2O 

Oxidation  of  Organic  Acids. — The  various  organic  acids  present  in 
animal  or  plant  residues,  or  produced  by  bacteria  in  the  course  of  the  de- 
composition of  carbohydrates,  alcohols,  and  other  carbonaceous  organic 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  127 


substances,  especially  if  the  oxidative  processes  are  restricted  by  the 
absence  of  air,  will  undergo  further  oxidation  in  the  presence  of  air 
under  the  influence  of  numerous  fungi  and  bacteria.  Free  acids  are  at- 
tacked mostly  by  fungi,  while  the  bacteria  generally  prefer  the  neutral 
salts.  The  following  table  gives  a summary  of  results  obtained  in  such 
experiments  d 


Decomposition 
Positive  ( + ) or 
Negative  ( — ) 

For- 

mic 

Acid 

Acetic 

Acid 

Pro- 

pionic 

Acid 

Lactic 

Acid 

Suc- 

cinic 

Acid 

Malic 

Acid 

Tar- 

taric 

Acid 

Citric 

Acid 

Bacterium  fluorescens 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Bacterium  prodigiosum. . . . 

+ 

- 

- 

- 

+ 

+ 

— 

-1- 

Bacterium  erythrogenes . . . 

- 

- 

- 

- 

- 

+ 

+ 

+ 

Bacterium  aerogenes 

+ 

- 

- 

+ 

+ 

+ 

— 

+ 

Bacterium  acidi  lactici 

+ 

+ 

- 

+ 

+ 

+ 

+ 

+ 

Bacterium  coli 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Bact.  vulgare  (Proteus).  . . 

+ 

- 

- 

- 

- 

+ 

+ 

+ 

Bacillus  subtilis 

- 

- 

- 

+ 

— 

+ 

- 

+ 

Bacillus  mesentericus 

+ 

+ 

+ 

+ 

+ 

+ 

-■ 

+ 

Oidium  lactis 

+ 

+ 

— 

— 

Even  such  a highly  resistant  acid  as  oxalic  acid  which  accumulates  in 
considerable  quantities  in  certain  plants,  for  instance  in  the  leaves  of 
sugar  beets,  can  be  oxidized  by  different  bacteria. 

Formation  and  Destruction  of  Humus. — The  decomposition  of 
organic  residues  gives  rise  not  only  to  numerous  chemically  well  defined 
substances,  but  also  to  relatively  complex  products  of  brown  or  black 
color,  collectively  called  humus.  Its  presence  is  very  conspicuous  in 
barnyard  manure  as  well  as  in  soil,  and  practical  experience  has  always 
indicated  that  there  is  a close  relationship  between  the  fertility  of  a given 
soil  and  its  humus  content.  The  colloidal  nature  of  the  humus  com- 
pounds enables  them  to  store  vast  quantities  of  plant  food  in  such  a man- 
ner that  it  is  easily  accessible  to  the  soil  organisms  as  well  as  to  the  roots 
of  the  cultivated  plants.  Nearly  all  nitrogen  present  in  soil  is  there  in 
the  form  of  humus  nitrogen ; the  gradual  decomposition  of  the  humus 
liberates  part  of  this  nitrogen  as  ammonia  which  is  quickly  nitrified.  Con- 
siderable quantities  of  carbon  dioxide  are  evolved  simultaneously,  which 
increase  the  solubility  of  the  mineral  constituents  of  the  soil.  Insofar  as 
the  carbon  dioxide  escapes  from  the  soil,  it  adds  to  the  supply  of  carbon 
dioxide  in  the  air  that  is  available  for  the  green  plants. 

The  dark  color  of  the  humus  compounds  is  often  accepted  as  indi- 

1 A.  Maassen,  Arb.  a.  d.  kais.  Gesundheitsaml,  vol.  12,  1S96,  p.  340-411. 


128 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


eating  a high  carbon  content  of  these  substances.  However,  only  in  cer- 
tain cases,  especially  in  peat,  is  the  percentage  of  carbon  distinctly  higher 
than  in  carbohydrates.  The  humus  of  rich  field  soils,  on  the  other  hand, 
is  usually  relatively  low  in  carbon,  but  comparatively  high  in  nitrogen, 
as  may  be  seen  from  the  following  data  d 


Percentage 

Carbon 

Nitrogen 

Peat  humus 

52-64 

0.5-4 

Field  humus 

30-56 

3-9 

The  relation  of  N : C in  green  plants  is  1 : 25-40,  hut  only  1 : 10-15  in  field 
humus.  This  difference  shows  that  relatively  more  of  the  nitrogenous 
plant  components  participate  in  humus  formation  than  do  the  carbon- 
aceous substances.  If  the  transformation  takes  place  under  anaerobic 
conditions,  as  in  peat  deposits,  more  carbon  is  retained,  while  in  well 
aerated  soils  more  carbon  dioxide  is  liberated  hv  oxidative  processes. 
The  rather  resistant  feces  of  many  large  and  small  animals  make  up  a 
large  part  of  the  humus  in  soil,  as  they  do  in  barnyard  manure.  But 
also  ammonia  as  well  as  certain  amino  acids  give  dark  humus-like  com- 
pounds when  combined  with  carbohydrates.2  When  so  much  ammonium 
carbonate  or  sulfate  (plus  chalk)  is  added  to  straw  that  the  mixture  con- 
tains 0.7  per  cent  N,  a darkly  colored  material  is  formed  within  8-12 
weeks,  which  looks  and  acts  very  similar  to  barnyard  manure,  and  no 
longer  exerts  such  detrimental  effects  in  soil  as  are  characteristic  of  fresh 
straw.3 

The  chemistry  of  humus  is  only  partly  known.  0.  Schreiner  and  his 
associates  were  able  to  isolate  from  different  soils  a large  number  of 
aliphatic  and  cyclic  compounds,  which  participate  in  humus  forma- 
tion.4 When  humus  is  purified  as  far  as  possible,  a distinctly  acid  char- 
acter is  noticeable,  and  it  is  these  humic  acids  that  are  partly  responsible 
for  the  sour  character  of  peaty  soils.  In  fertile  soil,  neutralization  takes 
place  by  combination  with  ammonia,  lime,  and  other  basic  substances,  and 
such  neutral  or  slightly  alkaline  humus  is  much  more  easily  oxidized  by 
fungi  and  bacteria,  as  well  as  by  purely  chemical  processes.  The  end 

1 F.  Lohnis,  “ Handbuch  der  landw.  Bakteriologie  ” 1910,  p.  550-554. 

2 Maillabd,  “ Genese  des  matieres  proteiques  et  des  matieres  humiques.”  Paris, 
1913,  p 301-396. 

3H.  B.  Hutchinson  and  E.  H.  Richards,  Jour.  Min.  Agr.,  Gr.  Britain,  vol.  2S, 
1921,  p.  398-411. 

4 E.  C.  Lathrop,  Journ.  Franklin  Inst.,  vol.  183,  1917,  p.  169;  J.  J.  Skinner,  1.  c.f 
vol.  186,  1918,  p.  165. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  129 


products  are  always  carbon  dioxide  and  water,  but  part  of  the  humic  sub- 
stances are  again  used  for  rebuilding  the  cells  of  the  active  microor- 
ganisms. 

Formation  and  Assimilation  of  Carbon  Dioxide. — The  decomposition 
of  humus  furnishes  most  of  the  carbon  dioxide  needed  by  the  green  plants 
for  assimilation.  Burning  of  wood  and  coal,  the  respiration  of  men,  ani- 
mals, and  plants,  as  well  as  volcanic  activities  produce  also  considerable 
quantities  of  carbon  dioxide.  But  in  view  of  the  fact  that  from  every 
acre  of  land  approximately  5000-10,000  lbs.  of  organic  matter  are  har- 
vested annually,  of  which  perhaps  two-third  remains  upon  or  returns  to 
the  soil  in  the  form  of  crop  residues,  green  manures,  and  straw  in  stable 
manures,  where  they  fall  prey  to  humification  and  carbon  dioxide  for- 
mation, it  becomes  evident  that  these  processes  are  of  superior  importance 
in  regard  to  the  continuous  production  of  food  for  men  and  animals. 
Free  air  contains  as  a rule  not  more  than  0.03  to  0.04  per  cent  carbon 
dioxide,  while  the  air  inclosed  in  fertile  soil  often  contains  0.5  to  3.0,  and 
occasionally  up  to  10  per  cent  or  more.  As  there  is  a continual  exchange 
of  oxygen  and  carbon  dioxide  between  atmosphere  and  soil,  the  carbon 
dioxide  of  the  soil  becomes  accessible  to  the  growing  plants,  whose  leaves 
are  turning  their  stomata,  as  a rule,  toward  the  soil.  There  is  no  doubt 
that  the  greater  supply  of  carbon  dioxide  furnished  by  a soil  rich  in 
neutral  humus,  is  largely  responsible  for  the  better  crops  obtainable  upon 
such  soils. 

Soil  organisms  may  also  assimilate  some  carbon  dioxide.  If  a rich 
flora  of  algae  is  present,  as  in  wet  soils,  the  effect  may  become  noticeable. 
Furthermore,  there  are  a few  groups  of  bacteria — the  nitrifying,  hydro- 
gen oxidizing,  certain  sulfur  and  iron  bacteria — that  are  also  able  to 
assimilate  carbon  dioxide.  But  these  activities  are  practically  quite  ir- 
relevant. The  nitrifying  bacteria,  which  are  most  active  in  soil,  assimilate 
only  1 part  of  carbon  while  oxidizing  35  to  40  parts  of  nitrogen.  In 
other  words,  if  70  to  80  lbs.  of  nitrogen  are  nitrified  in  one  acre  of  land, 
not  more  than  2 lbs.  of  carbon  are  assimilated,  which,  of  course,  is  of  no 
importance  whatever  when  compared  with  the  humus  present  in  the  soil 
and  with  the  quantities  of  carbon  added  in  green  and  stable  manures. 

Formation  and  Metabolism  of  Carbon  Monoxide,  Methane,  and 
Hydrogen. — Considerable  quantities  of  carbon  monoxide  are  liberated 
in  the  incomplete  combustion  of  coal.  Small  amounts  have  also  been 
found  in  stable  manure,  where  they  are  probably  produced  by  bacterial 
activity.  Nothing  definite  is  known  in  this  respect.  But  several  micro- 
organisms have  been  isolated  that  are  able  to  assimilate  and  to  oxidize 
carbon  monoxide,  despite  its  being  poisonous  to  all  other  organisms. 


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Most  common  among  them  is  Bac.  oligocarbopliilus,  first  studied  by 
Beijerinck. 

Large  quantities  of  methane  are  produced  in  the  absence  of  air  by 
numerous  bacteria  from  very  different  substances,  such  as  proteins, 
amino  acids,  carbohydrates,  organic  acids,  and  alcohols.  Gases  within 
the  intestinal  tract  and  in  barnyard  manure  contain  frequently  not  less 
than  50  per  cent  of  methane.  Soil  covered  by  water  shows  the  same  gas 
formation,  especially  if  it  is  rich  in  organic  substances.  In  well  aerated 
soils,  however,  very  little  methane  or  none  is  to  be  found.  A so- 
called  Bac.  methanicus  and  several  other  species  have  proved  themselves  ' 
capable  of  making  use  of  methane  for  assimilation  and  for  respiration. 

Hydrogen  is  produced  under  the  same  conditions  as  is  methane,  al- 
though usually  in  smaller  quantities.  Its  oxidation  occurs  partly  spon- 
taneously, partly  as  a result  of  the  activity  of  various  bacteria  that  make 
use  of  the  energy  obtained  for  the  assimilation  of  carbon  dioxide.  But 
this  metabolism  is  by  no  means  their  only  mode  of  life.  If  there  is  no 
hydrogen,  as  in  most  soils,  they  live,  like  other  microorganisms,  on  organic 
food,  and  they  can  therefore  not  be  properly  classed  as  a separate,  well 
defined  group  of  “autotrophic”  bacteria. 

5.  TRANSFORMATION  OF  MINERAL  SUBSTANCES 

Bacteria  and  fungi  are  responsible  for  most  of  the  transformations  of 
carbon  and  of  nitrogen.  In  regard  to  the  metabolism  of  the  so-called 
mineral  substances,  such  as  potassium,  calcium,  iron,  sulfur,  and  phos- 
phorus, bacterial  action  is  much  less  conspicuous,  although  by  no  means 
unimportant.  In  the  course  of  the  decomposition  of  organic  residues  these 
substances  are  attacked  by  the  same  microorganisms  which  break  down 
the  carbon  and  nitrogen  compounds.  Physical  and  chemical  factors,  on 
the  other  hand,  are  mostly  responsible  for  the  disintegration  of  rocks  and 
for  the  gradual  increase  in  solubility  of  the  mineral  soil  constituents. 
Carbon  dioxide  as  well  as  organic  and  inorganic  acids  are  active  in  these 
respects,  and  since  large  quantities  of  them  are  produced  by  microorgan- 
isms, the  latter  are  again  of  importance  although  in  an  indirect  manner. 
Soluble  minerals  are  used  by  bacteria  and  fungi  as  by  higher  organisms 
for  cell  construction.  The  phosphorus  requirement  of  many  bacteria  is 
relatively  large,  when  compared  with  that  of  cultivated  plants. 

Mineralization  of  Organic  Residues. — The  mineralization  of  organic 
residues  remains  always  more  or  less  incomplete.  Part  of  the  nitrogen 
and  carbon  is  assimilated  by  the  active  microorganisms,  and  also  a part 
of  the  phosphorus,  potassium,  calcium,  iron,  and  sulfur.  Rarely  more 
than  50  per  cent  of  the  nitrogen  of  barnyard  manure  is  nitrified  in  the 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  131 


soil  during  the  first  three  or  four  years.  Similar  relations  prevail  in  re- 
gard to  the  mineral  constituents  of  stable  manure,  although  the  increase 
in  solubility  is  usually  somewhat  greater  than  in  the  case  of  nitrogen. 
The  following  data  were  obtained  from  such  experiments  concerning  the 
percentage  of  nitrogen,  phosphorus,  and  potassium  recovered  in  the  crops 
grown : 


Percentage  Recovered 

Nitrogen 

Phosphorus 

Potassium 

Field  experiments,  2 years  1 

28-34 

25-34 

43-70 

3 years  2 

32-51 

30-41 

? 

4 years  3 

7-46 

10-76 

22-85 

Pot  experiments,  3 years  2 

54 

103 

? 

Similar  results  have  been  obtained,  but  within  a much  shorter  time, 
when  in  fish  guano  the  phosphorus  soluble  in  carbonated  water  was  de- 
termined, before  and  after  the  material  was  fed  to  animals.4  Originally 
not  more  than  18  per  cent  was  soluble;  in  the  feces  the  solubility  was 
increased  to  52  to  68  per  cent,  and  after  the  excreta  were  kept  for  two 
months  62  to  73  per  cent  of  the  phosphorus  had  been  transformed.  That 
in  certain  cases  the  opposite  reaction,  that  is,  the  assimilation  of  min- 
eral compounds  may  become  more  or  less  conspicuous,  is  illustrated,  for 
instance,  by  the  fact  that  phosphates  added  to  stable  manure  lost  within 
a relatively  short  time  24  to  64  per  cent  of  their  soluble  phosphorus.5 

Metabolism  of  Phosphorous  Compounds. — To  the  organic  phosphor- 
ous compounds  belong  the  nucleoproteids,  the  phytins,  and  the  phos- 
phatides  (lecithin),  of  which  the  latter,  as  a rule,  are  more  easily  de- 
composed than  the  former.  Inorganic  phosphates  are  frequent  in  soil, 
but  the  humus  contains  also  varying  amounts  of  organic  phosphorus. 
Plant  residues  (green  manure,  straw)  are  comparatively  rich  in  organic 
phosphorus,  especially  phytin;  animal  residues  (guano,  bone  meal)  are 
characterized  by  a relatively  high  content  of  phosphates.  Whether  there 
is  a conspicuous  change  from  the  inorganic  to  the  organic  form  or  vice 
versa,  is  largely  dependent  on  the  supply  of  bacterial  nutrients,  especially 
of  carbohydrates.  The  general  situation  is  similar  to  that  existing  in  the 
mineralization  or  assimilation  of  nitrogen  compounds.  The  presence  of 
carbohydrates  stimulates  assimilation  and  checks  mineralization ; lack  of 

3W.  Schneidewind,  Landw . Jahrb.,  vol.  39,  Erg.  Bd.  Ill,  1910,  pp.  62-74. 

2 B.  Welbel,  Travaux  du  Labor,  chimique  de  la  Stat.  agr.  de  Ploty,  1908,  pp.  58,  62. 

3B.  Schulze,  Arb.  Deutsch.  Landw.  Gesellsch.,  198,  1911,  pp.  167,  170,  174. 

4 0.  Kellner,  Landw.  Vers.  Stat.,  vol.  20,  1877,  p.  433. 

3 W.  E.  Tottingham  and  C.  Hoffmann,  Wis.  Agr.  Exp,  Stat.  Research  Bull.  29, 1912. 


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carbohydrates  exerts  the  opposite  influence.  Humus  furnishes  food  for 
the  assimilating  microorganisms  and  fixes  at  the  same  time  part  of  the 
phosphorus,  as  well  as  the  ammonia,  by  absorption.  The  detailed  study 
of  these  processes  meets  with  difficulties,  because  the  sterilization  of  a 
soil  changes  its  absorptive  power  in  a varying  degree,  and  a smaller  or 
larger  part  of  the  phosphorus  is  usually  so  firmly  fixed  by  absorption  that 
its  solubility  is  not  very  different  from  that  of  the  phosphorus  which  was 
assimilated  by  soil  organisms. 

Bacterial  Action  upon  Phosphates. — Some  decades  ago,  when  raw 
bone  meal  was  used  as  fertilizer,  it  was  often  mixed  with  animal  manures 
and  kept  for  several  weeks  before  being  applied.  The  increased  efficiency 
of  such  “fermented”  material  was  thought  to  be  due  to  a better  solubility 
of  the  phosphates,  while  in  fact  the  ammonification  of  organic  nitrogenous 
compounds  and  the  removal  of  fatty  substances  were  undoubtedly  of 
much  greater  influence.  It  goes  without  saying  that  in  all  cases  where 
the  alkalinity  is  increased  by  ammonification  very  little  dissolu- 
tion of  the  phosphate  can  be  expected.  A prompt  reaction  is  possible 
only  where  acids  are  present  or  are  formed  which  transform  the  triphos- 
phates into  di-  and  mono-phosphates.  Raw  rock  phosphates  are  therefore 
of  value  in  distinctly  acid  soils  and  in  prairieland  where  an  exceptionally 
high  humus  content  assures  a vigorous  foi’mation  of  organic  acids  and  of 
large  quantities  of  carbon  dioxide.  Composting  of  rock  phosphates  with 
sulfur  and  soil  can  also  serve  as  a means  of  increasing  the  solubility  of 
the  phosphates,  provided  that  the  bacterial  formation  of  sulfuric  acid 
proceeds  vigorously  enough  to  effect  a quick  transformation  of  the  phos- 
phates.1 As  a rule,  the  direct  application  of  acid  phosphate  will  be  pref- 
erable. Nitrification  is  practically  without  effect  upon  the  solubility  of 
the  phosphates  in  soil.  The  quantity  of  nitrous  and  nitric  acid  formed  is 
so  small  compared  with  the  basic  substances  present  in  an  average  soil, 
that  hardly  any  free  acid  will  have  an  opportunity  to  attack  the  phos- 
phates.2 The  action  of  carbon  dioxide  is  undoubtedly  of  greater  effect 
in  all  soils  which  contain  enough  humus. 

Bacterial  Action  upon  Carbonates  and  Silicates. — The  transforma- 
tion of  alkali  and  calcium  carbonates  and  silicates  shows  many  analogies 
to  the  metabolism  of  phosphorus  compounds.  The  indirect  effects  exerted 
by  microorganisms  are  again  of  greater  importance  than  their  direct  ac- 
tions. Carbon  dioxide,  organic  acids,  nitric  and  sulfuric  acids  increase 
the  solubility  of  the  carbonates  and  silicates.  As  a rule  carbon  dioxide 

' J.  G.  Lipman  et  ah,  Soil  Science,  vol.  5, 1918,  pp.  243-250;  vol.  11, 1921,  pp.  87-92. 

’ J.  W.  Ames  and  T.  E.  Richmond,  Soil  Science,  vol.  6,  1918,  pp.  351-364;  W.  P. 
Kelley,  Jour.  Agric.  Research,  vol.  12,  1918,  pp.  671-683. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  133 


takes  the  first  rank.  It  is  well  known  that  feldspar,  mica,  and  similar 
minerals  are  very  resistant,  and  special  experiments  made  with  the  in- 
tention to  secure  a quicker  disintegration  by  bacterial  activity  did  not 
furnish  any  remarkable  results.1  Other  less  resistant  silicates  of  potassium, 
such  as  are  present  in  the  so-called  green  sands  of  New  Jersey  and  Mary- 
land, were  made  somewhat  more  soluble  when  they  were  composted  with 
sulfur  and  manure,2  but  also  in  this  case  an  economic  advantage  is  hardly 
to  be  expected. 

A few  exceptions  are  known  where  acids  produced  by  bacteria  have 
proved  able  to  perform  a strong  solvent  action.  One  of  them  is  the 
gradual  disintegration  of  a mountain  peak  in  Switzerland  (the  so-called 
Faulhorn  in  the  Berner  Oberland)  which  is  caused  by  excessive  nitrifi- 
cation, due  to  an  exceptionally  high  nitrogen  content  of  the  rocks.  A 
rapid  deterioration  of  concrete  structures  has  taken  place  in  some  cases 
when  they  were  immersed  in  water  rich  in  sulfuric  acid  or  sulfates.  Like- 


Fig.  33. — Sulfur  cycle. 


wise,  organic  acids  have  repeatedly  caused  serious  damage  by  their  de- 
structive actions  in  dairies  as  well  as  in  silos. 

Sulfur  Metabolism. — The  transformation  of  sulfur  and  sulfur  com- 
pounds shows  many  parallelisms  to  the  cycle  of  niti’ogen,  as  may  be  seen 
from  a comparison  of  Fig.  33  with  Fig.  29  (p.  96).  In  both  cases  the 
decomposition  of  the  organic  compounds  leads  at  first  to  the  formation 
of  hydrogen  compounds  (NH3  and  SH2),  which  are  then  oxidized  to 
nitric  and  to  sulfuric  acid,  respectively.  A marked  difference  exists  in- 
, sofar  as  elementary  sulfur,  instead  of  nitrous  acid,  appears  as  an  inter- 
mediate step  in  this  process,  according  to  the  following  formulae: 

2H2S+02  = 2H20+S2 
2HoO+S2+3  02  = 2H2S04 

1 Bassalik,  Zeitschr.f.  Garungsphysiol .,  vol.  2,  1912,  pp.  1-32;  vol.  3,  1913,  pp.  15-42. 

5 McCall  and  A.  M.  Smith,  Jour.  Agric.  Research , vol.  19,  1920,  pp.  239-256. 


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Furthermore,  it  is  to  be  pointed  out  that  while  sharply  differentiated 
groups  of  microorganisms  are  active  in  the  processes  of  ammonifieation 
and  nitrification,  the  same  bacteria  are  often  able  to  produce  hydrogen 
sulfide  as  well  as  sulfuric  acid.  In  the  absence  of  air  the  organic  sulfur 
is  mostly  converted  into  hydrogen  sulfide,  but  intensive  aeration  stimu- 
lates direct  oxidation  to  sulfuric  acid,  which  is  immediately  changed  to 
sulfates. 

The  retrograde  processes  display  also  many  parallelisms.  Sulfates 
as  well  as  free  sulfur  may  be  reduced  to  hydrogen  sulfide ; sulfates  and 
hydrogen  sulfide  may  be  assimilated ; free  sulfur  may  be  liberated  from 
hydrogen  sulfide  as  well  as  from  sulfates.  The  thiosulfates  which  are 
produced  by  spontaneous  oxidation  of  hydrogen  sulfide  and  of  other  sul- 
fides undergo  analogous  reactions.  They  may  be  reduced  to  hydrogen 
sulfide  or  oxidized  to  sulfates ; at  the  same  time  elementary  sulfur  may 
be  precipated  and  part,  of  the  sulfur  may  be  used  in  the  process  of  assimi- 
lation. 

Purely  chemical  reactions  play  a much  more  important  role  in  the 
transformation  of  sulfur  and  sulfur  compounds  than  they  do  in  the  cycle 
of  nitrogen.  Hydrogen  in  the  nascent  state,  as  it  occurs  in  many  fer- 
mentative processes,  may  produce  hydrogen  sulfide  from  organic  sulfur 
compounds,  from  elementary  sulfur,  from  sulfites,  or  from  thiosulfates. 
Sulfate  reduction  alone  seems  to  be  exclusively  due  to  bacterial  action, 
whereas  the  oxidation  of  hydrogen  sulfide  and  of  free  sulfur  to  sulfates  is 
again  caused  by  microorganisms  as  well  as  by  purely  chemical  reactions. 

Formation  of  Hydrogen  Sulfide. — Nearly  all  of  the  very  numerous 
bacteria  and  fungi  that  are  able  to  produce  ammonia  from  organic  sub- 
stances have  also  been  found  to  be  connected  with  the  production  of 
hydrogen  sulfide  or  of  ammonium  sulfide  from  proteins.  This  process 
occurs  in  the  presence  as  well  as  in  the  absence  of  air,  at  low  as  well  as 
at  high  temperatures.  Spoiled  eggs  are  often,  though  not  always,  char- 
acterized by  the  liberated  hydrogen  sulfide  or  ammonium  sulfide.  The 
flavor  of  certain  cheeses,  such  as  Limburger,  is  partly  due  to  hydrogen 
sulfide  or  ammonium  sulfide;  if  traces  of  metal  have  entered  such  curd 
a gray  or  black  discoloration  of  the  cheese  may  become  noticeable.  Oc- 
casionally some  sulfur  may  get  into  the  milk  from  improperly  vulcanized 
rubber  tubes  used  on  milking  machines ; the  hydrogen  sulfide  produced 
causes  a marked  deterioration  of  the  flavor  of  such  milk,  especially  if 
this  is  kept  at  a relatively  high  temperature. 

The  offensive  odors  characteristic  of  putrid  substances  are  probably 
always  partly  due  to  the  presence  of  hydrogen  sulfide  and  of  ammonium 
sulfide,  but  volatile  organic  sulfur  compounds,  such  as  mercaptane,  may 
contribute  to  the  effect.  Mercaptane  is  a by-product  of  the  metabolism 


vpmrij 


Lohnis-Fred,  Text  book 


Plate  IX 


2.  Iron  bacteria 

2/3  nat.  size 


1.  Sulfur  bacteria 

2/3  nat.  size 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  135 


of  many  bacteria  that  produce  hydrogen  sulfide;  it  is  further  split  by 
them  into  alcohol  and  hydrogen  sulfide. 

Formation  of  Sulfates. — Organic  sulfur  compounds,  hydrogen  sul- 
fide, thiosulfates,  and  sulfur  may  all  be  oxidized  to  sulfates.1  Abundant 
aeration  induces  many  bacteria  active  in  the  decomposition  of  proteins, 
to  transform  the  organic  sulfur  directly  to  sulfates  instead  of  hydro- 
gen sulfide.  Hydrogen  sulfide  and  other  sulfides  are  equally  attacked  by 
numerous  oxidizing  bacteria,  while  thiosulfates  and  elementary  sulfur 
are  oxidized  by  only  comparatively  few  species. 

Compounds  rich  in  oxygen,  such  as  nitrates,  can  support  these  oxida- 
tions in  the  absence  of  free  oxygen,  as  illustrated  in  the  formula  : 

12  KNOa+5  S2+4  CaC03  = 6 K2SO4+4  CaS04+4  C02+6  N2. 

Denitrification  takes  place  in  this  case  in  the  absence  of  organic  sub- 
stances ; the  active  species  has  been  named  Thiobacillus  denitrificans. 

If  mud  rich  in  hydrogen  sulfide,  usually  blackened  by  the  sulfides 
which  it  contains,  is  kept  under  a layer  of  water  of  moderate  depth,  fre- 
quently the  interesting  phenomenon  pictured  in  Plate  IX  becomes  visible. 
Provided  that  the  water  in  the  cylinder  is  kept  perfectly  quiet  and  is 
protected  from  the  light,  the  oxidizing  bacteria  will  accumulate  as  a 
“plate”  or  “niveau”  at  a point  in  the  solution  where  they  get  enough 
hydrogen  sulfide  from  below  and  enough  oxygen  from  above.  The  ap- 
pendices visible  below  the  small  funnel-shaped  depressions  in  the  plate 
are  formed  by  the  motile  bacteria  diving  down  to  gather  the  hydrogen 
sulfide  and  returning  to  the  level  where  the  oxidation  is  perfected.  If 
the  depth  of  the  water  were  reduced  the  bacteria  would  return  into  the 
mud  at  the  bottom,  and  the  oxidation  of  the  sulfides  would  become  visible 
by  a change  in  the  color  from  black  to  gray. 

Sulfur  Bacteria. — Although  all  bacteria  connected  with  the  metabol- 
ism of  sulfur  are  properly  called  sulfur  bacteria,  this  designation  is  fre- 
quently used  in  a more  restricted  sense  for  those  bacteria  only  which 
oxidize  hydrogen  sulfide,  sulfur,  and  thiosulfates  to  sulfates.  The  term 
thiosulfate  bacteria  is  also  applied  to  the  last-named  group  of  organisms, 
but  they,  too,  are  able  to  oxidize  sulfur  as  well  as  hydrogen  sulfide.  As 
mentioned  above,  many  species  are  known  to  be  members  of  this  group, 
and  in  regard  to  their  morphology  they  display  as  many  differences  as 
have  been  observed  with  other  bacteria. 

The  term  “ sulfofication  ” is  sometimes  used  to  designate  these  processes,  but  it  is 
as  incorrectly  chosen  as  were  simitar  terms  (azofication  and  rhizofication)  mentioned 
on  pp.  97  and  98.  “Sulfofication’’  would  mean  formation,  not  oxidation,  of  sulfur. 


136 


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Figure  34  shows  a picture  as  it  is  often  seen  when  a drop  of  water 
containing  sulfur  bacteria  is  examined  under  the  microscope.  Most  of  the 
cells  visible  are  filled  with  minute  droplets  of  col- 
loidal sulfur,  which  is  deposited  within  the  cells 
as  an  intermediate  product  of  the  oxidation  of 
hydrogen  sulfide  to  sulfate.  This  accumulation  of 
finely  divided  sulfur  causes  the  whitish  color  of 
the  plate  of  sulfur  bacteria,  as  shown  in  Plate  IX. 
Long  threadlike  forms  (Beggiatoa,  Thiothrix,  and 
others)  as  well  as  short,  mostly  motile,  rod-like  and 
Fig.  34.— Sulfur  bacteria  sPiral  cells  are  almost  invariably  to  be  found.  If 

living.  X1000.  such  mud  water  is  exposed  to  the  sun-light,  a 
luxuriant  growth  of  purple-colored  bacteria  fre- 
quently becomes  established.  These  organisms  are  in  part  of  very  large 
dimensions  and  otherwise  of  peculiar  appearance.1 

As  causative  agents  of  the  oxidation  of  thiosulfates  and  of  sulfur  two 
species  seem  to  be  of  greatest  importance,  that  is,  Thiobacillus  thioparus 
and  Thiobacillus  thiooxidans,  the  latter  being  of  special  interest  because 
of  its  inclination  to  grow  in  distinctly  acid  substrates  and  to  produce 
and  to  withstand  hydrogen-ion  concentrations  down  to  pH=0.6.2  The 
oxidation  of  sulfur  in  phosphate-sulfur  composts,  mentioned  on  p.  132, 
is  mainly  due  to  its  activity.  When  thiosulfates  are  oxidized,  frequently 
part  of  the  sulfur  is  temporarily  deposited  outside  of,  not  within  the 
cells,  as  in  the  case  of  Beggiatoa  and  other  species  oxidizing  hydrogen 
sulfide.  The  reaction  proceeds  in  the  following  manner : 

2 Na2S203+02  = 2 Xa2S04-|-S2. 

Part  of  the  sulfur  bacteria  (Beggiatoa,  Thiobacillus,  and  some  others) 
make  use  of  the  energy  liberated  in  the  sulfate  formation,  for  the  assimi- 
lation of  carbon  dioxide,  just  as  is  done  by  the  nitrifying  bacteria  in  the 
oxidation  of  ammonia  to  nitrite  and  nitrate.  Even  the  relations  between 
nitrogen  or  sulfur  oxidized  and  carbon  assimilated  are  the  same  in  both 
cases  (40:1),  as  was  ascertained  in  regard  to  sulfur  oxidation  by  Thio- 
bacillus.3 

Sulfate  Reduction. — The  production  of  hydrogen  sulfide  from  ele- 
mentary sulfur  is  very  common  among  bacteria  and  fungi,  but  the  retro- 
grade transformation  of  sulfates  to  hydrogen  sulfide  has  thus  far  been 

1 H.  Molisch,  “ Die  Purpurbakterien,”  Jena,  1906. 

2 S.  A.  Waksman  and  J.  S.  Joffe,  Jour.  Bad.,  vol.  7,  1922,  pp.  239-256. 

3 Lieske,  Sitzgs.-Ber.  Akad.  Heidelberg,  Mathem.-naturw.  Kl.,  (B)  1912,  6.  Abhand- 
lung. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  137 


observed  with  only  two  anaerobic  spirilla.  If  gypsum  is  added  to  barn- 
yard manure  a veiy  intensive  reduction  may  become  noticeable.  Analo- 
gous changes  may  occur  in  ponds,  water  reservoirs,  and  also  in  water- 
logged soils.  Since  the  roots  of  most  of  the  cultivated  plants  are  very 
sensitive  to  hydrogen  sulfide,  proper  aeration  of  field  soils  becomes  of  im- 
portance on  this  account,  too. 

Iron  Metabolism. — Considerable  quantities  of  iron  are  present  in  the 
form  of  carbonates  or  organic  salts  in  the  water  circulating  in  the  soil. 
To  a lesser  degree  the  same  holds  true  in  regard  to  manganese.  By  spon- 
taneous oxidation  of  the  bicarbonates  the  hydroxides  of  iron  and  man- 
ganese may  be  deposited,  and  part  of  the  precipitates  accumulating  in 
water  drains,  conduits,  ditches,  as  well  as  in  the  soil,  are  undoubtedly 
formed  in  this  way. 

However,  numerous  microorganisms  are  able  to  act  in  an  analogous 
manner.1  Clogging  of  water  pipes  by  iron  deposits  has  repeatedly  been 
found  to  be  due  to  the  abundant  growth  of  iron  bacteria.  These  organ- 
isms make  use  either  of  the  carbon  dioxide  of  the  carbonates,  or  of  the 
organic  acids  of  the  iron  and  manganese  salts.  The  first  group  repre- 
sents another  type  of  carbon  dioxide  assimilating  microorganisms.  The 
hydroxides  of  iron  and  manganese  accumulate  in  smaller  or  larger  quan- 
tities in  colloidal  form  in  the  slimy  capsules  or  sheaths  of  the  iron 
bacteria.  Later  a slow  crystallization  takes  place  which  destroys  the  soft 
bacterial  cells  and  leaves  the  so-called  bog  ore,  which  presents  in  all  re- 
spects the  appearance  of  a material  of  purely  mineral  origin.  Soils  rich 
in  humus  may  also  contain  an  amount  of  hydroxides  of  iron  and  man- 
ganese held  in  suspension  by  the  protective  action  of  humus  colloids. 
When  the  latter  are  destroyed  by  microorganisms,  the  hydroxides  are 
precipitated  upon  the  active  cells. 

Production  of  inorganic  and  of  organic  acids  by  bacteria  and  fungi 
may  lead,  on  the  other  hand,  to  increased  solubility  of  iron  and  man- 
ganese in  the  soil,  and  simultaneously  to  an  increased  iron  content  of  the 
drainage  water.  Alfalfa  and  some  other  crops  which  leave  large  quan- 
tities of  stubble  and  roots  in  the  soil  exert  occasionally  a very  marked  in- 
fluence in  this  respect,  because  a more  active  decomposition  takes  place 
under  such  conditions. 

Iron  Bacteria. — Drainage  water  of  peat  soil  is,  as  a rule,  exception- 
ally rich  in  iron  bacteria,  which  not  only  form  brown  deposits  at  the 
bottom  of  the  ditches,  but  also  accumulate  in  thin  iridescent  films  on  the 
surface  of  the  water,  where  such  accumulations  have  sometimes  been  mis- 

1 H.  Molisch,  “Die  Eisenbakterien,”  1910;  E.  C.  Harder,  U.  S.  Geological  Survey, 
Professional  Paper  113,  1919. 


138 


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taken  for  oil.  In  Plate  IX  a cylinder  is  shown  partly  filled  with  swampy 
soil  and  water  in  which  an  iron  rod  has  been  immersed.  As  far  as 
oxygen  has  permeated  the  water,  the  iron  bacteria  have  settled  upon 
the  iron  rod  as  well  as  on  the  side  of  the  glass,  and 
the  characteristic  film  upon  the  surface  has  also 
been  produced.  Figure  35  demonstrates  how  these 
organisms  appear  when  examined  microscopically. 
Some  of  them  are  comparatively  large;  Crenothrix, 
Gallionella,  and  Leptothrix  belong  to  this  group. 
Numerous  other  species  of  average  size  and  shape 
participate  in  these  processes,  as  do  even  fungi 
and  algae.  As  mentioned  above,  manganese  may 
replace  iron,  and  the  oxidation  of  ferrous  bicar- 
bonate enables  some  of  the  iron  bacteria  to  as- 
similate carbon  dioxide. 

6.  PATHOGENIC  ACTION  OF  MICROORGANISMS 

Compared  with  the  very  large  number  of  microorganisms  continually 
active  in  nature  as  “mediators  between  death  and  life”  of  the  higher 
organisms,  there  are  only  comparatively  few  species  that  are  inclined  to 
enter  into  close  relations  with  higher  plants  and  animals  either  on  a 
symbiotic  or  on  an  antagonistic  basis.  The  last-named  group  comprises 
all  the  pathogenic  bacteria,  whose  intimate  study  represents  the  domain 
of  medical  bacteriology.  Investigation  and  discussion  of  the  infectious 
diseases  of  plants,  animals,  and  men  are  the  objects  of  plant  pathology, 
and  of  veterinary  and  human  medicine,  as  was  pointed  out  on  pp.  1 and  2. 
However,  a general  review  of  the  activities  of  bacteria  would  be  incom- 
plete if  the  fundamental  facts  concerning  the  pathogenic  action  of  micro- 
organisms were  entirely  omitted.  Therefore  a brief  discussion  of  these 
general  principles  will  be  given  on  the  following  pages. 

Resistance  Against  Infectious  Diseases. — As  indicated  by  the  term 
antagonistic  action,1  it  is  the  struggle  between  the  invading  bacteria  and 
the  invaded  organism  that  is  characteristic  of  all  infectious  diseases. 
Either  the  bacteria  are  victorious  and  the  higher  organism  succumbs,  or 
the  latter  overcomes  and  kills  the  bacteria.  This  double-faced  character 
of  the  problem  is  frequently  overlooked.  Especially  during  the  first 
decades  after  the  discovery  of  the  causative  agents  of  anthrax,  cholera, 
typhoid,  tuberculosis,  etc.,  the  bacteriological  standpoint  was  often  too 
much  emphasized.  Many  people  have  lived  and  still  live  in  permanent 
fear  that,  they  may  come  in  contact  with  these  dangerous  bacteria.  But 


Fig.  35. — Iron  bacteria 
living.  X1000. 


'Derived  from  the  Greek  word  avraywricrTlis  (antagonist es)  = opponent. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  139 


increased  knowledge  of  bacterial  action  was  accompanied  by  a growing 
insight  into  the  various  ways  by  which  a healthy  body  offers  a more  or 
less  successful  resistance  against  infectious  diseases.  The  hygienic  point 
of  view  was  thus  brought  to  the  foreground,  and  the  belief  was  and  is 
sometimes  held  that  under  proper  hygienic  conditions  any  action  of  the 
pathogenic  bacteria  is  forestalled  because  of  the  resistance  offered  by  a 
healthy  body. 

It  need  hardly  be  emphasized  that  both  extreme  views  are  not  tenable. 
A large  quantity  of  highly  pathogenic  bacteria  introduced  into  a suscep- 
tible organism  will  promptly  cause  the  disease,  while  this  will  not  happen 
if  only  a few  bacterial  cells  of  reduced  pathogenicity  invade  the  host. 
The  resistance  offered  by  a healthy  body  is  undoubtedly  very  great,  but 
because  no  organism  is  continually  in  a state  of  perfect  health,  chances 
for  falling  sick  are  not  infrequent.  Therefore,  the  bacteriological  and  the 
hygienic  points  of  view  are  of  equal  importance,  although  for  practical 
reasons  the  latter  may  take  first  rank,  as  is  expressed  by  the  proverb : 
Prevention  is  better  than  cure. 

Bacterial  Pathog’enicity. — In  earlier  times  when  pathogenic  bacteria 
were  still  unknown  there  was  a widespread  belief  that  contagious  diseases 
were  closely  related  to  putrefactive  processes.  This  assumption  was  un- 
doubtedly correct  insofar  as  in  such  cases  where  proteins  are  decom- 
posed by  bacteria  extremely  poisonous  substances — so-called  ptomaines1 
— may  be  formed,  which  are  capable  of  causing  deadly  diseases.  Spoiled 
meat,  cheese,  and  other  decomposed  foods  rich  in  proteins  may  owe  their 
more  or  less  poisonous  character  to  the  presence  of  ptomaines.  More 
frequently,  however,  a certain  poison  produced  by  a species  called  B. 
botulinus  is  responsible  for  food  poisoning.  Improperly  treated  silage 
as  well  as  imperfectly  sterilized  food  may  contain  this  agent  of  botulism, 
which  causes  every  year  a considerable  number  of  fatal  accidents. 

Other  poisons,  usually  called  toxins,  are  produced  by  the  majority  of 
pathogenic  bacteria.  In  certain  cases,  however,  the  luxuriant  growth  of 
the  microorganisms  alone  is  sufficient  to  lead  to  serious  disturbances  in 
the  host  ’s  metabolism,  either  by  clogging  part  of  the  blood  vessels  or  by 
otherwise  interfering  with  the  normal  chemical  processes. 

The  effects  exerted  by  pathogenic  bacteria  are  not  always  character- 
ized by  a very  distinct,  sharply  defined  and  localized  disease.  Not  infre- 
quently different  and  variable  symptoms  may  become  noticeable  and  the 
pathogenicity  itself  may  be  more  or  less  reduced.  There  are,  in  fact, 
many  indications  that  the  pathogenic  bacteria  are  related  to  non-patho- 
genic  forms,  and  it  is  quite  probable  that  in  the  different  stages  of  their 


1 Derived  from  the  Greek  word  irriD/ia  (ptoma)  = corpse 


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development  the  pathogenicity,  too,  may  vary  widely.  The  sudden  ap- 
pearance and  disappearance  of  epidemics,  which  is  often  very  con- 
spicuous, seems  to  have  its  main  cause  in  these  as  yet  little  known  fea- 
tures of  the  development  of  these  organisms.1 

Virulence  and  Infection. — Although  bacterial  pathogenicity  is  not 
invariably  due  to  toxin  production,  it  has  nevertheless  become  a general 
usage  to  consider  pathogenicity  and  virulence  synonymous  terms.2 
Strains  are  called  highly  virulent  if  their  pathogenicity  is  at  its  height, 
and  avirulent  when  they  are  no  longer  able  to  produce  a disease. 
Avirulent  strains  are  rather  frequent  with  the  relatively  common  infec- 
tious diseases,  such  as  typhoid,  diphtheria,  and  tuberculosis.  But  these 
so-called  pseudo-diphtheria  and  pseudo-tuberculosis  bacteria  are  also  of 
great  pathological  interest,  because  they  may  regain  their  virulence 
under  conditions  not  yet  well  known. 

When  pathogenic  bacteria  invade  an  organism  it  depends  on  their 
virulence  and  on  the  resistance  offered  by  the  invaded  body  whether  or 
not  an  infection  will  take  place.  If  a disease  is  caused,  but  the  organism 
recuperates,  the  virulent  bacteria  may  be  retained  in  the  body  for  a short, 
and  sometimes  for  a long  period.  Such  apparently  healthy  “carriers” 
of  a disease  are  not  uncommon,  especially  with  typhoid,  diphtheria,  and 
cholera.  Because  the  bacteria  are  still  virulent  in  these  cases,  such  car- 
riers are  liable  to  become  a permanent  danger  to  unprotected  individuals. 

The  time  which  elapses  until  invasion  is  followed  by  infection  is 
called  the  period  of  incubation ,3  Cell  multiplication  and  toxin  produc- 
tion takes  place  during  this  period  until  sufficient  quantities  of  infective 
material  are  produced. 

Endo-  and  Ecto-toxins. — Endo-  and  ecto-enzymes  play  prominent 
roles  in  the  metabolism  of  higher  as  well  as  of  lower  organisms.  In  the 
same  manner  the  toxins  produced  by  the  bacterial  cells  are  either  re- 
tained therein  as  endo-toxins,  or  they  leave  the  cells  and  act  as  so-called 
ecto-toxins.  Generally  the  latter  are  more  active  than  the  former. 
The  high  mortality  rate  in  fully  developed  cases  of  diphtheria  and  of 
tetanus  is  due  to  the  presence  of  such  ecto-toxins  which  quickly  poison 
the  whole  organism.  As  in  the  case  of  the  botulinus  toxin  mentioned 
above,  heat,  light,  and  various  chemical  substances  can  reduce  or  destroy 
the  efficiency  of  these  ecto-toxins. 

The  endo-toxins,  obtained  in  the  form  of  extracts  from  the  cells  by 
applying  high  pressure  or  by  grinding  the  previously  dried  bacteria,  do 
not  act  in  such  a specific  manner  as  do  the  ecto-toxins.  The  living  bac- 

1 Almquist,  Jour.  Infect.  Diseases, ~v ol.  31,  1922,  no.  5,  p.  483. 

2 Virulence  is  derived  from  the  Latin  word  virus  = poison. 

3 Derived  from  the  Latin  word  incubare  = brood,  hatch. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  141 


teria,  however,  act  differently,  and  this  may  be  due  to  their  specific  be- 
havior within  the  body,  as  well  as  to  the  production  of  specific  substances 
not  yet  well  known.  The  so-called  aggressins  belong  to  this  group ; they 
have  no  marked  toxic  properties,  but  they  stimulate  the  efficiency  of  the 
pathogenic  bacteria  very  distinctly. 

Immunity  and  Immunization. — If  the  resistance  offered  by  an  or- 
ganism against  a disease  is  sufficiently  strong  to  destroy  and  to  eliminate 
the  pathogenic  bacteria  and  to  keep  the  body  healthy,  the  organism  is 
called  immune  against  that  kind  of  disease.  If  all  individuals  of  the 
same  species  are  immune  against  a disease,  the  immunity  is  absolute,  but 
it  is  relative  if  only  certain  individuals  prove  to  be  resistant.  Absolutely 
immune,  for  instance,  are  men  against  Rinderpest,  cattle  against  glanders, 
most  animals  against  typhoid.  The  relative  immunity  is  strongly  influ- 
enced by  the  age  and  the  general  living  conditions  of  the  individual; 
adverse  factors  (hunger,  cold,  fatigue)  may  impair  the  disease  resistance 
seriously.  The  immunity  may  be  inherited,  or  it  may  be  later  acquired 
either  actively  or  passively,  that  is,  either  by  having  once  been  exposed 
to  the  disease,  or  by  having  been  protected  against  it  by  direct  immuniza- 
tion (application  of  immune  serum  or  chemical  treatment). 

The  reaction  of  the  immune  organism  against  bacterial  infection 
must  be  twofold ; the  bacteria  themselves  must  be  killed  and  eliminated, 
and  their  toxins  must  be  neutralized.  If  non-toxic  bacteria  invade  an 
organism,  as  frequently  happens  with  common  non-pathogenic  bacteria 
getting  into  lesions  of  the  outer  skin  or  of  the  inner  lining  of  the  body, 
the  bactericidal  action  is  very  prompt.  Because  of  the  adverse  environ- 
mental conditions  the  bacteria  die  within  a short  time  and  are  speedily 
digested  by  enzymes  of  the  blood.  This  prompt  bactericidal  action  tends 
to  keep  the  blood  and  inner  organs  of  the  animal  body  free  from  bacteria 
despite  the  frequent  chances  for  contamination.  If  toxins  are  produced 
by  the  bacteria,  the  immune  organism  checks  their  effect  by  the  produc- 
tion of  so-called  antitoxins,  that  are  specific  anti-bodies  present  in  and 
transferable  with  the  blood  or  the  blood-serum  of  the  immune  organism. 

Phagocytosis. — The  fight  against  and  the  elimination  of  pathogenic 
bacteria  is  performed  either  by  bacteriophagous  cells  or  by  specifically 
acting  substances  in  the  blood-serum  (see  bacteriolysis).  The  blood  con- 
tains red  corpuscles  and  white  cells,  so-called  leucocytes.  As  was  first 
observed  by  Metchnikoff,  the  latter  are  able  to  devour  and  to  digest 
pathogenic  bacteria,  acting  in  this  case  as  so-called  phagocytes } When 
examined  microscopically  such  cells  display  a striking  resemblance  to 
certain  protozoa  (compare  Fig.  36  with  Fig.  16,  Plate  I,  and  Text -Fig. 


1 Derived  from  the  Greek  word  <paydv  (phagein)  = devour. 


142 


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56).  However,  phagocytosis  does  not  always  proceed  in  a prompt  and 
speedy  manner.  Sometimes  the  phagocytes  seem  to  be  paralyzed  and 
unable  to  attack  the  bacteria.  The  bacterial  aggressins  mentioned  above 
are  accepted  as  cause  of  this  inactivity,  but  their  paralytic  action  can  be 
overcome  by  an  adequate  counteraction  of  the  infected  body.  Specific 
antiaggressins  are  produced  by  the  blood-serum,  which  are  usually 
called  opsonins,1  in  order  to  indicate  that  they  exert  a stimulating  effect 
upon  the  inclination  of  the  phagocytes  to  ingest  and  to  digest  the 
aggressin  producing  bacteria. 

Bacteriolysis  and  Agglutination.— If  the  blood  of  an  immune  organ- 
ism is  freed  from  its  phagocytes  and  the  serum  alone  is  allowed  to  act 
upon  the  bacteria,  again  a bactericidal  and  bacteriolytic  action  may  be- 
come noticeable,  for  which  several  substances  have  been  made  responsible. 
Some  of  them  are  produced  by  the  phagocytes,  while  others,  so-called 


abed 

Fig.  36. — Phagocytes  after  Metchnikoff  (a-c)  and  Bordet  (d).  a-b.  Anthrax  bacilli; 
c.  Streptococci;  d.  Epithelioma  contagiosum. 


alexins,2  are  normal  components  of  the  immune  serum.  It  was  mentioned 
on  p.  56  that  those  secreta  of  the  leucocytes  have  been  connected  with 
the  recently  discovered  bacteriophagy  in  the  intestines  (d’Herelle’s 
phenomenon),  although  it  is  well  beyond  doubt  that  in  this  as  in  other 
cases  where  bacteriophagy  was  observed  outside  of  the  host,  unknown 
substances  or  organisms  are  the  cause  of  this  antagonistic  effect. 

When  bacteria  are  tested  in  blood-serum  not  always  a genuine  bac- 
teriolysis takes  place ; the  bacteria  are  not  promptly  killed  and  dissolved, 
but  merely  agglomerated  and  precipitated.  This  reaction,  the  so-called 
agglutination,  is  probably  of  no  importance  for  the  infected  organism, 
but  it  is  of  considerable  value  for  certain  diagnostic  purposes.  In  cases 
of  typhoid,  for  instance,  the  agglutination  test  (Widal’s  reaction)  has 
proved  very  helpful.  Even  the  identification  of  non-pathogenic  bacteria 
is  frequently  based  upon  the  results  of  analogous  tests.  By  inoculating 

1 Derived  from  the  Greek  word  Bfov  (opson)  = seasoning,  relish. 

2 Derived  from  the  Greek  word  &\££eiv  (alexein)  =ward  off. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  143 

a certain  strain  of  bacteria  into  the  blood  vessels  of  an  animal,  an  immune 
serum  can  be  obtained  which  agglutinates  this  as  well  as  closely  related 
strains  of  bacteria.  But  occasionally  very  erratic  results  are  obtained.1 
For  instance,  serum  of  an  animal  immunized  against  tetanus  agglutinated 
typhoid  bacteria  more  vigorously  than  B.  tetani ; another  serum 
expected  to  act  specifically  against  mold  spores,  proved  again  more  effect- 
ive against  typhoid  bacteria.  Therefore,  too  much  stress  should  not  be 
laid  upon  the  outcome  of  such  agglutination  tests. 

Hemolysis. — If  instead  of  bacteria,  blood  of  another  organism  is 
inoculated  into  an  animal,  again  anti-bodies  appear  in  the  serum  which 
exert  in  this  case  a lytic  action  upon  the  red  corpuscles  of  the  foreign 
blood.  These  hemolysins  may  be  used  to  ascertain  experimentally 
whether  blood  of  unknown  origin  is  that  of  man  or  of  some  kind  of  ani- 
mal, and  so  to  decide  a question  which  in  murder  trials  sometimes  be- 
comes of  great  importance.  Analogous  reactions  are  possible  against 
many  other  substances,  but  of  special  interest  is  a peculiar  test  that  is 
based  upon  the  combined  action  of  bacteriolysins  and  hemolysins  in  so- 
called  inactivated  and  reactivated  sera.  The  mechanism  of  this  test  is  as 
follows : 

Moderate  heating  makes  a serum  “inactive,”  that  is,  its  bacteriolytic 
or  hemolytic  action  is  paralyzed ; but  it  will  be  reactivated  if  a small 
amount  of  fresh  serum  is  added.  The  substances  which  were  destroyed 
by  heating  and  are  introduced  again  with  the  fresh  serum  are  called  com- 
plements, because  the  action  of  bacteriolysins  and  hemolysins  will  be  com- 
plete only  if  these  complements  are  present.  If  blood  is  mixed  with 
inactivated  hemolytic  serum  and  bacteria  are  added  in  inactivated  bac- 
teriolytic serum,  at  first,  of  course,  no  reaction  can  take  place  in  the  mix- 
ture on  account  of  the  absence  of  complements.  As  soon  as  these  are 
added,  however,  they  will  activate  either  the  bacteriolysins  or  the  hemo- 
lysins of  the  mixed  sera ; if  the  bacteria  present  are  identical  with  those 
which  were  used  for  preparing  the  bacteriolytic  serum,  bacteriolysis  will 
take  place  and  no  hemolysis,  which  becomes  visible  only  if  the  bacteria  are 
of  another  kind.  This  very  valuable  test  has  been  invented  by  two  Bel- 
gian bacteriologists,  Bordet  and  Gengou ; in  its  application  upon  the  diag- 
nosis of  syphilis  it  has  become  known  universally  as  the  so-called  Wasser- 
mann  test.  In  doubtful  cases  of  cholera,  typhoid,  etc.,  it  can  also  be 
used  to  advantage. 

Vaccination. — It  was  mentioned  above  that  active  immunization 
against  an  infection  may  have  been  acquired  by  previous  exposure  to  the 

1 Lehmann  und  Neumann,  “Grundriss  der  Bakteriologie,”  5 Aufl.,  1912,  p.  135; 
Kruse,  “ Allgemeine  Mikrobiologie,”  1910,  p.  1088. 


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same  disease.  In  cases  of  measles,  scarlet  fever,  whooping  cough,  for 
instance,  the  immunity  acquired  in  early  childhood  is  usually  sufficient 
for  the  rest  of  the  life.  In  other  diseases,  as  in  foot  and  mouth  disease, 
the  immunity  obtained  will  persist  only  for  a short  period,  and  there 
are  still  other  cases  (influenza,  gonorrhea)  where  practically  no  immunity 
against  a new  infection  is  secured.  But  wherever  active  immunization 
occurs,  the  possibility  exists,  as  was  discovered  by  Edward  Jenner  in 
regard  to  smallpox,  that  the  immunization  acquired  from  a relatively 
mild  form  of  the  disease  will  also  afford  protection  against  its  more  viru- 
lent forms.  The  inoculation  of  the  vaccin  taken  from  calves  produces,  as 
a rule,  a mild  local  affliction  which,  however,  creates  an  active  immunity 
against  the  much  more  dangerous  smallpox,  and  this  immunity  will  last 
for  a number  of  years.  Since  it  is  relatively  easy  to  reduce  experiment- 
ally the  virulence  of  pure  cultures  of  pathogenic  bacteria  to  any  desired 
degree,  all  kinds  of  vaccins  can  be  prepared  in  the  laboratories,  and  some 
of  them  have  proved  very  helpful  in  combating  infectious  diseases. 
Anthrax,  for  instance,  was  formerly  one  of  the  most  dreaded  animal 
diseases,  because  the  spores  of  B.  anfhracis  remain  alive  in  and  on  the 
soil  for  decades  and  are  liable  to  cause  new  infections  continually  after 
one  has  occurred  in  a locality,  but  it  has  now  lost  much  of  its  terror  since 
Pasteur  discovered  a vaccination  which  has  been  much  improved  during 
the  last  thirty  years. 

Serum  Treatment. — It  takes  time,  of  course,  before  an  active  im- 
munization can  be  perfected  by  vaccination,  and  this  is  therefore  of  value 
only  as  a prophylactic  treatment.  But  in  certain  cases  it  is  possible  to 
vaccinate  experimental  animals  with  gradually  increased  doses  of  bac- 
teria or  of  toxins,  until  anti-bodies  are  formed  in  their  blood  in  such  large 
quantities  that  a comparatively  small  amount  of  the  blood-serum  car- 
ries enough  protective  substances  to  exert  a prompt  curative  effect,  if  it 
is  applied  before  the  disease  has  been  firmly  established.  This  type  of 
passive  immunization  has  become  of  greatest  importance  and  has  proved 
highly  beneficial  in  the  treatment  of  diphtheria.  Some  other  diseases 
are  accessible  to  an  analogous  treatment,  but  the  great  hopes  which 
followed  Behring’s  discovery  of  the  serum  therapy  of  diphtheria  were 
premature  and  exaggerated.  The  protection  afforded  by  passive  im- 
munization can  be  completed  by  combining  serum  treatment  with  vac- 
cination. 

A peculiar  fact  that  plays  an  important  role  in  the  preparation  as 
well  as  in  the  application  of  immune  serum  is  the  so-called  anaphylactic 
reaction  of  the  treated  organism.  The  term  anaphylaxis1  was  chosen  in 

1 Derived  from  the  Greek  words  avb  (ana)  = upward,  and  d(pu\aKTos  (aphvlaktos)  = 
unguarded,  defenceless;  that  is,  anaphylaxis  = increased  defencelessness. 


ACTIVITIES  OF  BACTERIA  AND  RELATED  MICROORGANISMS  145 


order  to  indicate  that  for  some  time  after  the  first  injection  was  made, 
an  increased  susceptibility  is  noticeable.  If  a first  vaccination  is  quickly 
followed  by  a second  injection,  the  anaphylactic  shock  produced  may 
kill  the  experimental  animal,  but  if  this  does  not  happen,  anti-anaphyl- 
axis is  reached  and  further  injections  will  do  no  harm. 

Chemotherapy. — For  a considerable  length  of  time  the  interest  of 
medical  bacteriologists  was  concentrated  almost  exclusively  upon  vaccina- 
tion and  serum  treatment.  But  it  goes  without  saying  that  in  the 
struggle  against  pathogenic  bacteria  the  use  of  chemical  substances  may 
also  be  of  great  benefit.  Mercury  and  arsenic  compounds  have  proved 
very  valuable  in  the  treatment  of  syphilis,  the  application  of  chaul- 
moogra  oil  promises  to  eradicate  leprosy,  which  has  long  been  considered 
incurable.  It  is  to  be  hoped  that  an  equally  effective  treatment  will  be 
found  against  tuberculosis,  since  extensive  experiments  to  produce  active 
immunization  by  vaccination  have  not  been  successful.  Other  diseases, 
too,  may  be  more  accessible  to  chemotherapy  than  to  vaccination  and 
serum  treatment.  It  depends  on  the  circumstances  which  of  the  three 
methods  will  give  the  best  results. 


Part  II 


DAIRY  AND  SOIL  BACTERIOLOGY 


# 


CHAPTER  VIII 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  FOODSTUFFS 

As  was  pointed  out  in  Chapter  IV,  5,  bacteria  and  related  microor- 
ganisms accompany  the  cycle  of  matter  that  starts  from  and  returns  to 
the  soil.  Originally  all  microorganisms  come  from  the  soil,  but  the 
changes  in  environmental  conditions  cause  numerous  quantitative  modi- 
fications in  the  microflora,  as  is  found  in  foodstuffs,  dairy  produce, 
manure,  etc.  It  was  also  mentioned  before  (p.  63)  that  all  growing 
plants  are  covered  by  an  almost  continuous  layer  of  bacteria  specifically 
adapted  to  their  habitat ; in  addition  they  are  more  or  less  contaminated 
by  dirt,  dust,  and  manure.  The  different  ways  in  which  foodstuffs  are 
treated  after  having  been  harvested,  lead  necessarily  to  further  altera- 
tions in  the  composition  of  the  microflora  in  regard  to  number  as  well  as 
kind  of  organisms  present. 

Germ  Content  of  Foodstuffs. — When  seeds  are  planted  in  sterilized 
soil  rapid  multiplication  of  the  relatively  few  bacteria  that  stick  to 
every  seed  starts  as  soon  as  germination  takes  place,  and  the  young 
sprouts  are  being  covered  by  the  slimy  layer  of  bacteria  that  is  charac- 
teristic of  all  green  plants.  The  following  counts  exemplify  these  rela- 
tions d 

Number  of  bacteria  ( 011  each  seed  planted 3,000-S0,000 

\ on  young  sprouts  grown 750,000-19,750,000 

Molds  and  bacteria  may  be  found  not  only  on  the  outside,  but  also  in 
the  inner  parts  of  seeds.  Leguminous  seeds  especially  are  sometimes 
heavily  infested,  as  is  indicated  by  their  inability  to  develop  normal 
sprouts  in  germination  tests.  In  other  cases  the  microorganisms  within 
the  seeds  are  of  importance  as  symbionts,  as  was  mentioned  on  p.  113. 

The  number  of  microorganisms  present  upon  the  different  parts  of 
full  grown  plants  varies,  of  course,  within  wide  limits  according  to  plant 
species,  conditions  of  growth,  contaminations  by  dust,  etc.  The  different 

1 Complete  references  relating  to  dairy  and  soil  bacteriology  are  given  in  the  senior 
author’s  “Handbuch  der  landwirtschaftlichen  Bakteriologie,”  as  far  as  they  were 
published  before  autumn  1910.  Additional  summaries  are  printed  in  the  Zeitsch.  f. 
Garungsphysiologie,  vol.  1,  1912,  pp.  68,  340,  and  in  the  Centralbl.  f.  Bakt.,  II.  Abt., 
vol.  54,  1921,  p.  273. 


149 


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treatment  given  to  hay,  silage,  straw,  and  gi’ain  causes  additional  modi- 
fications. However,  it  is  not  so  much  the  presence  of  microorganisms  as 
it  is  their  activities  that  are  of  real  interest.  It  will  suffice  to  give  a few 
data  showing  the  minimal  and  maximal  counts  of  bacteria  and  fungi  per 
gram. 


Green  forage 2,000,000-200,000,000 

Hay 7,000,000-  17,000,000 

Straw 10,000,000-400,000,000 

Grain  (cereals) 100,000-  12,000,000 

Concentrated  feed  (oil  cakes,  etc.) 10,000-  20,000,000 

Microflora  of  Different  Foodstuffs. — Various  organisms  are  growing 
in  the  slimy  bacterial  layer  that  is  characteristic  of  the  epidermis  of  green 
plants.  Most  common  among  them  are  Bad.  fluorescens  and  a species 
called  Bad.  herbicola;  the  latter  produces  a yellow  or  reddish 
pigment  that  is  sometimes  visible  with  the  naked  eye  on  young  sprouts, 
especially  on  fresh  barley  malt  kept  under  humid  conditions.1  Plants 
which  are  supplied  with  stable  manure  are  usually  contaminated  by  fecal 
lactic  acid  bacteria  ( B . coli  and  aerogenes ) ; if  human  feces  were  applied, 
pathogenic  organisms,  such  as  B.  typhosus,  may  be  found  occasionally. 
Lactic  acid  streptococci  and  lactobac-illi  are,  as  a rule,  comparatively 

rare  on  growing  plants,  but  on  cabbage  and  corn  these  organisms  are 

more  frequent,  and  these  two  plants  are,  therefore,  especially  inclined 
to  undergo  an  acid  fermentation  in  the  sauerkraut  vat  or  in  the  silo. 
Flax,  hemp,  and  other  fiber  plants  have  also  a peculiar  microflora ; bac- 
teria and  fungi  attacking  pectic  substances  are  rather  numerous  in  such 
cases.  Spores  of  the  common  soil  bacteria,  such  as  B.  sub  tills,  mesenteri- 
cus  and  amylobader,  are  carried  by  the  dust  as  contaminations.  Their 
presence  and  resistance  make  a complete  sterilization  of  green  vegetables 
sometimes  difficult.  Potatoes  and  beet-roots  are,  of  course,  exposed  to 
very  heavy  contaminations  by  such  organisms,  whose  growth  may  cause 
serious  losses,  if  temperature  and  humidity  are  high  in  the  places 
of  storage.  Still  more  accidental  and  irregular  is  the  microflora  of  con- 
centrated feeding  stuffs.  Some  authors  were  of  the  opinion  that  bac- 
teriological tests  would  allow  of  an  accurate  judgment  in  regard  to  the 
quality  of  such  material,  but  the  tests  made  did  not  confirm  this  hy- 
pothesis. 

G rass,  hay,  and  straw  contain  almost  regularly,  though  not  in  great 
numbers,  various  representatives  of  a group  of  bacilli  related  to  B.  tuber- 
culosis. Some  of  them  have  been  explicitly  named  “grass  bacilli”  or 
“timothy  bacilli.”  When  found  in  milk,  butter,  and  cheese,  they  have 

1 H.  T.  Gussow,  Canada  Expt.  Farms  Report,  1911,  p.  241. 


Bacteria  and  related  microorganisms  in  foodstuffs  151 


been  repeatedly  mistaken  for  true  tubercle  bacilli.  In  their  typical  form 
they  are  not  pathogenic  for  men,  but  their  virulence  can  be  increased, 
and  their  general  character  may  be  so  changed  experimentally  that  they 
assume  practically  all  the  features  of  the  tubercle  bacillus.1  However, 
under  natural  conditions  this  transformation  will  not  often  take  place. 

Hay  Bacteria. — When  grass  is  made  into  hay,  part  of  the  bacteria 
will  die,  but  slime  production  and  spore  formation  enable  many  of  them 
to  remain  alive  although  in  a dormant  state.  If  the  weather  is  favorable 
or  the  drying  is  done  artificially,  not  many  chemical  alterations  will  take 
place.  Under  less  favorable  conditions,  however,  far-reaching  changes 
may  occur,  and  the  nutritive  value  of  the  hay  will  be  more  or  less  im- 
paired. As  long  as  the  water  content  of  the  material  is  not  too  low,  the 
cell  enzymes  remain  active,  and  the  respiration  of  the  cells  goes  on,  re- 
sulting in  a loss  of  approximately  10  per  cent  of  the  organic  substances.2 
The  specific  aroma  production  is  due  to  the  splitting  of  certain  glucosides. 
Part  of  the  proteins  (10  to  40  per  cent)  are  transformed  into  amides 
and  amino  acids.  The  reduction  in  the  percentage  of  carbohydrates, 
fats,  and  organic  phosphorus  compounds  ranged,  according  to  weather 
conditions,  within  the  following  limits : 3 


Per  Cent 

Sucrose 22-87 

Glucose 27-88 

Starch 2-28 


Per  Cent 


Dextrin 0-45 

Fats 10-40 

Phosphorus  compounds 7-29 


Phosphates  increased  accordingly,  while  cellulose  as  well  as  pectic  sub- 
stances remained  unchanged  as  long  as  practically  no  bacterial  activity 
took  place.  Unfavorable  weather,  however,  stimulates  unavoidably  the 
growth  of  bacteria  and  molds,  and  their  destructive  activities  become 
sometimes  very  marked,  especially  when  clover  or  alfalfa  is  made  into 
hay. 

The  so-called  hay  bacillus,  Bac.  subtilis,  as  well  as  other  sporulating 
strains  that  are  related  to  Bac.  mesentericus,  the  so-called  potato  bacillus, 
can  be  easily  brought  to  good  development  if  hay  is  placed  in  water  and 
the  mixture  boiled  for  a few  minutes.  After  a few  days  the  liquid  is 
covered  with  a whitish  film  characteristic  of  these  organisms. 

Bacterial  Activities  in  Stored  Hay. — While  hay  making  tends  to 
suppress  all  bacterial  activity,  and  this  aim  is,  in  fact,  reached  if  the 
weather  is  not  too  unfavorable,  bacteria  will  always  become  active  again 

1 W.  Kolle,  H.  Schlossberger  und  W.  Pfannstiel,  Deutsche  Medic.  Wochenschr., 
vol.  47,  1921,  p.  437. 

2 F.  Fleischmantst,  Landw.  Vers.  Stat.,  vol.  76,  1912,  pp.  237-447. 

3 Fleischmann,  1.  c. 


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after  the  hay  is  stored,  and  the  so-called  sweating  process  sets  in.  The 
heat  and  moisture  appearing  in  the  hay  stack  at  this  time  are  in  part  the 
result  of  the  respiration  of  surviving  plant  cells,  but  the  increase  in 
temperature  and  humidity  favors  the  development  of  bacteria  and  fungi, 
which  in  turn  add  to  the  effect  by  their  respiration.  Under  normal 
conditions  all  these  activities  keep  within  comparatively  narrow  limits. 
The  rapid  bacterial  multiplication  is  followed  by  an  equally  sudden  de- 
crease in  numbers,  and  after  four  to  six  weeks  comparatively  few 
vegetative  cells  together  with  more  numerous  spores  may  be  found.  Diig- 
geli  obtained,  for  instance,  the  following  counts  in  millions  per  g.  hay: 

First  Day  Seventh  Day  Fourteenth  Day 

18  2,400  6 

If  the  material  was  originally  rich  in  B.  coli,  this  species  is  especially 
liable  to  multiply  rapidly,  and  together  with  some  other  bacteria  it  is 
probably  the  cause  of  the  harmful  effects  repeatedly  observed  when  such 
sweating  hay  was  fed  to  horses  before  its  “auto-sterilization”  was  com- 
plete. 

The  addition  of  1 per  cent  salt  proves  helpful  in  suppressing  ex- 
cessive bacterial  and  mold  growth  in  hay  harvested  in  wet  weather.  If 
too  much  heat  is  generated  in  the  hay  stack,  which  may  eventually  lead 
to  spontaneous  ignition,  as  was  discussed  in  Chapter  VII,  1,  brine  or 
compressed  carbon  dioxide  should  be  brought  to  the  danger  spots  by 
means  of  gas  pipes  or  similar  appliances.  The  taking  down  of  an  en- 
dangered hay  stack,  which  is  often  recommended,  is  highly  dangerous 
because  the  access  of  air  increases  the  chances  for  a sudden  ignition. 
It  should  never  be  attempted  unless  plenty  of  water  is  available. 

Sometimes  hay  is  stacked  while  its  water  content  is  still  comparatively 
high  (approximately  45  per  cent),  and  so-called  brown  bay  is  made. 
Enzymes  of  the  plant  cells  and  microorganisms  combine  their  effects; 
20  to  30  per  cent  of  the  organic  substances  are  destroyed;  proteins  are 
reduced  to  amino  acids  and  ammonia ; and  carbohydrates  are  changed  to 
organic  acids,  alcohols,  aldehyds,  and  carbon  dioxide.  This  acid  produc- 
tion together  with  the  high  temperature  checks  the  bacterial  activities, 
and  a fairly  valuable  feeding  stuff  is  obtained,  although  in  most  cases 
silage  is  superior,  because  here  the  unavoidable  losses  can  be  kept  at 
a much  lower  level. 

Making  of  Silage. — In  Europe  it  has  been  known  for  centuries  that 
it  is  possible  to  store  plants  rich  in  water  and  in  carbohydrates  in  the 
absence  of  air  without  considerable  losses,  and  that  within  a few  weeks 
they  are  transformed  into  a palatable  slightly  acid  product  that  keeps 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  FOODSTUFFS  153 


well  and  represents  a valuable  winter  forage.  In  the  first  decades  of 
the  19th  century  many  German  and  Hungarian  farmers  began  to  put 
part  of  their  forage  crops  into  pit  silos,  and  in  the  sixties  of  the  last 
century  Reihlen  of  Wiirttemberg  started  the  ensiling  of  maize  with 
very  satisfactory  results.1  In  1870  his  reports  were  translated  into 
French,  and  from  there  the  knowledge  of  this  process  came  to  America, 
where  I.  P.  Roberts  in  New  York,  F.  Morris  in  Maryland,  M.  Miles  in 
Illinois  and  W.  A.  Henry  in  Wisconsin  made  the  first  silage.  F.  H.  King 
also  in  Wisconsin  carried  out  the  first  careful  studies  of  the  process  and 
developed  the  American  style  of  silo  and  ensiling.  Recently  the  American 
system  has  been  introduced  into  European  farming,  while  the  old 
European  pit  silos  have  come  into  use  and  have  been  improved  upon  in 
the  Western  part  of  America.2 

At  first  it  was  thought  necessary  that  the  temperature  in  the  en- 
siled fodder  should  rise  to  50°  C.  in  order  to  get  silage  of  good  quality, 
and  this  point  is  again  frequently  discussed  in  the  more  recent  Euro- 
pean literature.  It  was  and  is  assumed  that  at  these  relatively  high  tem- 
perature a rapid  development  of  favorable  microorganisms  takes 
place,  and  thereby  the  detrimental  action  of  other  bacteria  is  inhibited. 
This  belief,  however,  is  not  well  founded.  The  most  important  point  is 
that  by  tight  packing  of  the  fodder  the  air  be  excluded  as  completely  as 
possible.  If  this  is  done  the  temperature  frequently  does  not  exceed 
30°  C.,  and  yet  silage  of  perfect  quality  is  obtained.  Next  to  tight  pack- 
ing, the  chemical  composition  of  the  ensiled  material  is  of  greatest  im- 
portance. When  the  fodder  is  too  young,  that  is,  if  it  contains  too  much 
protein  and  water,  a disagreeably  smelling,  unpalatable  product  will  re- 
sult. A relatively  large  percentage  of  carbohydrates  must  be  present, 
and  not  more  water  than  is  needed  to  make  juice  enough  to  fill  all  air 
spaces  between  the  fodder.  Com  and  sorghum  are  generally  best  suited 
for  silage,  because  they  are  not  too  rich  in  proteins.  Sunflowers,  sweet 
clover,  and  a mixture  of  oats,  vetch  and  peas  or  soy  beans  give  also  satis- 
factory results.  It  is  much  more  difficult  to  turn  clover  and  alfalfa  into 
good  silage ; their  moisture  content  is  usually  too  high.  The  rapid  forma- 
tion of  relatively  large  quantities  of  lactic  acid  is  essential  for  securing 
good  silage.  If  not  enough  lactic  acid  is  present,  butyric  acid  and 
disagreeably  smelling  products  of  the  protein  decomposition  come  to 
the  foreground ; such  silage  is  of  very  inferior  quality  or  a total  loss. 
The  following  data  3 may  indicate  how  the  acid  content  of  silage  varies 
(percentage  calculated  on  the  dry  weight  basis)  : 

1 L.  Carrier,  Jour.  Amer.  Soc.  Agron.,  vol.  12,  1920,  pp.  175-182. 

i U.  S.  Dept,  of  Agr.  Farmers’  Bull.  825,  1917. 

* R.  E.  Neidig,  Jour.  Agric.  Research,  vol.  14,  1918,  pp.  395-409 


154 


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Silage  Made  From 

Acetic 

Acid 

Propionic 

Acid 

Butyric 

Acid 

Lactic 

Acid 

Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

Corn 

1.8— 4.0 

0.2— 0.3 

0.0 

4.0— 6.1 

Sunflowers  

1.1— 3.4 

0.0 — 0.4 

0.0 — 3.1 

1 .5 — 3 5 

Oats  and  peas 

1.5— 2.2 

0.1— 0.2 

0.0 

4.6— 5.0 

Clover  and  straw 

2.0— 2.5 

0.2 

0.0 

2.8— 2.9 

Alfalfa  and  straw 

1.4— 2.0 

0.2— 1.0 

1.2— 2.2 

0.0 

The  making  of  good  silage  does  not  cause  greater  losses  than  approxi- 
mately 10  per  cent  of  the  nutritive  substances,  which  is  no  more  than  is 
lost  when  hay  is  made  under  favorable  circumstances. 


Fig.  37. — Acid-forming  bacteria  and  acid  production  in  corn  silage,  after  Esten  and 

Mason.1 


Microflora  of  Silage. — Immediately  after  the  fodder  is  put  into  the 
silo  a rapid  multiplication  of  bacteria,  especially  of  acid  producing 
species,  takes  place;  1000  millions  per  gram  may  be  frequently  found. 
But  this  rapid  increase  is  followed  by  an  equally  rapid  decrease,  and 

1 Esten  and  Mason,  Storrs  Agr.  Exper.  Stat.  Bull.  70.,  1912. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  FOODSTUFFS  155 


after  2 to  4 weeks  relatively  few  organisms  are  still  alive;  usually 
the  lactobaeilli  are  most  persistent.  The  acid  production  proceeds  more 
slowly  than  does  the  bacterial  growth,  as  is  shown  in  Fig.  37. 

There  has  been  much  discussion  in  the  literature  whether  the  cell 
enzymes  of  the  ensiled  fodder  or  the  microorganisms  growing  in  the 
silage  are  of  greater  importance.  It  can  not  be  doubted  that  the  cell 
enzymes  continue  to  work  after  the  material  is  put  into  the  silo,  but  it  is 
equally  certain  that  bacteria  also  play  an  important  part  in  the  forma- 
tion of  silage.  Lactic  acid  bacteria,  streptococci  as  well  as  lactobaeilli, 
are  numerous  in  good  silage.  Some  of  the  latter  reduce  fi’uetose  to 
mannitol,1  and  it  is  due  to  their  activity  that  silage  is  comparatively  rich 
in  this  substance.2  As  usual,  the  lactobaeilli  are  accompanied  by  yeasts ; 
both  groups  of  organisms  transform  part  of  the  glucose  to  alcohol 
which  is  of  influence  upon  the  flavor  of  good  silage.  Part  of  the  lactates 
first  formed  are  later  converted  into  acetates,  and  therefore  the 
flavor  of  old  silage  is  more  pungent  and  sour.  The  lactobaeilli  them- 
selves can  participate  in  this  secondary  process ; one  of  them,  called 
Lactobacillus  pentoaceticus,  produced,  for  instance,  in  young  cultures 
only  1 part  acetic  acid  to  every  8 parts  of  lactic  acid,  but  in  old  cultures 
the  relation  was  1 : 2,  because  of  the  transformation  of  lactates.3  Car- 
bon dioxide  and  alcohol  are  likewise  produced  by  lactobaeilli  as  well  as 
by  cell  enzymes.  Occasionally  carbon  dioxide  is  evolved  in  such  large 
quantities  that  it  becomes  dangerous  to  enter  the  silo,  because  of  lack  of 
oxygen  for  respiration. 

Treatment  of  Silage. — If  the  material  used  for  silage  is  naturally 
rich  in  carbohydrates,  in  plant  enzymes,  and  in  lactic  acid  bacteria,  as 
is  the  case  with  com,  no  other  treatment  is  needed  than  the  exclusion 
of  air  by  tight  packing.  Material  rich  in  protein  must  always  be  mixed 
with  substances  rich  in  carbohydrates,  such  as  corn  meal,  straw,  or  mo- 
lasses, and  the  addition  of  1 per  cent  salt  helps  to  suppress  the  protein 
decomposition  without  interfering  with  the  acid  formation.  If  there  is 
a lack  of  active  plant  enzymes  as  well  as  of  lactic  acid  bacteria,  as  is  the 
case  when  silage  is  made  from  boiled  potatoes,  or  from  the  waste  products 
of  beet  sugar  factories,  the  addition  of  active  lactic  acid  bacteria  is 
advisable  and  profitable.  The  use  of  such  so-called  pulp  cultures  origi- 
nated in  France  and  has  spread  to  all  European  countries  where  the 
beet  sugar  industry  furnishes  much  material  for  the  pit  silos.  The 
photograph  of  one  of  the  earliest  pulp  cultures,  manufactured  in  Austria, 

'E.  B.  Fred,  W.  H.  Peterson  and  J.  A.  Anderson,  Jour.  Biol.  Chem.,  vol.  58, 
1921,  p.  385, 

2 Dox  and  Plaisance,  Science  46,  1917,  p.  192. 

J W.  H.  Peterson  and  E.  B.  Fred,  Jour.  Biol.  Chem.,  vol.  42,  1920,  p.  278. 


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is  shown  in  Fig.  38.  If  such  residues  are  not  inoculated  usually  a 
vigorous  formation  of  butyric  acid  takes  place,  because  beets  as  well 
as  beet  tops  are  strongly  contaminated  by  these  common  soil  organisms 
whose  spores  survive  the  high  temperatures  applied  in  the  sugar  fac- 
tories. Beet  tops  should  always  be  kept  as  clean  as  possible.  Heavily 
soiled  material  can  never  be  made  into  good  silage;  putrefaction  and 
butyric  acid  formation  will  predominate,  and  the  silage  produced  will 
be  either  very  inferior  and  dangerous,  or  a complete  loss. 

During  the  last  few  years  an  electrical  treatment  of  silage  has  been 


Fig.  38. — Pulp  culture  manufactured  in  Vienna,  Austria,  with  directions  (t  nat.  size). 

9 

developed  in  Switzerland1  that  is  now  being  tested  extensively  in  different 
European  countries.  The  results  obtained  are  promising  as  far  as 
the  quality  of  the  material  is  concerned.  The  passing  of  the  electric  cur- 
rent raises  the  temperature  within  the  silo  quickly  to  about  50°  C.,  at 
which  temperature  only  plant  enzymes  and  certain  lactobacilli  display 
still  enough  activity  to  produce  the  necessary  acidity.  The  fermentative 
processes,  and  therefore  the  losses  of  nutritive  substances,  are  materially 
reduced,  but  it  remains  to  be  seen  whether  this  advantage  is  great  enough 
to  justify  the  relatively  high  costs  of  the  electrical  treatment ; about  1 
kilowatt  is  required  for  every  100  lbs.  of  fodder.  Several  authors  have 
1 Th.  Schweizer,  Deutsche  landw.  Presse,  vol.  48,  1921,  p.  343. 


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BACTERIA  AND  RELATED  MICROORGANISMS  IN  FOODSTUFFS  157 


asserted  that  all  bacteria  were  killed  by  the  electric  current  sent  through 
the  silage,  but  this  statement  is  without  foundation. 

Sauerkraut. — The  preservation  of  cabbage  by  spontaneous  acid 
fermentation  is  practically  the  counterpart  of  the  making  of  silage.  It 
is  very  probable  that  the  manufacture  of  sauerkraut,  as  practiced  in 
Europe  for  centuries,  has  been  the  basis  upon  which  all  similar  methods 
have  been  evolved.  Cabbage,  like  corn,  contains  lactic  acid  bacteria  which 
exert  a very  marked  influence  upon  the  quality  of  the  product,  although 
plant  enzymes  again  participate  in  the  process.  The  addition  of  salt  to 
the  cut  cabbage,  when  it  is  being  packed,  acts  favorably  in  two  directions. 
It  extracts  by  osmotic  action  the  sap  from  the  cells,  which  offers  itself 
as  a very  suitable  substrate  for  the  lactic  acid  bacteria,  and  simultaneously 
keeps  the  air  away  from  the  submerged  cabbage.  Furthermore,  many 
of  the  competitors  of  the  lactic  acid  bacteria  are  checked,  while  the  latter 
are  little  hindered  by  salt  concentrations  up  to  3 per  cent  or  more ; and 
taste  as  well  as  flavor  of  the  product  is  improved  by  the  predominance 
of  streptococci  and  lactobacilli.  Yeasts,  as  the  habitual  symbionts  of  the 
lactobacilli,  are  regularly  present  in  the  sauerkraut  vat  and  are  partly 
responsible  for  the  gas  formation  occuring  therein.  Occasionally  pink 
yeasts  may  become  so  numerous  that  kraut  of  pinkish  color  and  unde- 
sirable taste  is  produced.1 

Other  vegetables,  such  as  cucumbers,  green  beans,  etc.,  can  be  pre- 
served in  a similar  manner,  although  with  beans  failures  are  not  infre- 
quent due  to  their  higher  protein  content  and  a different  microflora, 
wherein  the  lactic  acid  bacteria  are  not  as  predominant  as  they  are  on 
cabbage.  Fresh  juice  from  sauerkraut  may  be  used  for  inoculation, 
while  sour  milk  or  whey  are  less  suitable  for  this  purpose ; the  lactic  acid 
bacteria  in  the  latter  case  being  mostly  adapted  to  milk  sugar,  not  to 
the  glucose  present  in  vegetables. 

Spoilage  of  Foodstuffs. — If  the  acid  formation  in  silage  remains  low, 
the  chances  for  spoilage  are  great,  and  it  depends  on  the  microflora  acci- 
dentally present  to  what  extent  this  will  take  place.  Usually  butyric 
acid  formation  and  putrefactive  processes  will  prevail,  and  the 
flavor  of  the  material  produced  will  be  so  disagreeable  that  the  animals 
will  refuse  to  eat  it.  Sometimes,  however,  it  may  happen  that  the  fodder 
was  contaminated  by  the  spores  of  B.  botulinus,2  and  such  silage 
may  become  very  dangerous,  like  the  incompletely  sterilized  vegetables 
mentioned  on  p.  139,  unless  the  germination  of  the  botulinus  spores  is 
forestalled  by  prompt  acidification. 

'E.  B.  Fred  and  W.  H.  Peterson,  Jour.  Bad.,  vol.  7,  1922,  p.  257. 

1 J.  S.  Buckley  and  L.  P.  Shippen,  Jour.  Amer.  Vet.  Assoc.,  vol.  50,  1917,  p.  809. 


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Stored  potatoes  and  beets  are  likewise  exposed  to  the  attacks  of  vari- 
ous bacilli  and  molds  which  may  cause  serious  losses.  If  the  tubers  and 
roots  are  diseased  when  they  are  placed  in  storage,  further  deterioration 
can  hardly  be  avoided.  Healthy  material,  however,  becomes  subject  to 
attacks  only  if  parts  of  its  tissues  are  first  killed  by  high  temperatures  and 
excessive  humidity,  or  by  frost.  Brown  spots  appearing  in  the  white 
tissue  of  freshly  cut  potatoes  indicate  dead  parts  of  the  tubers.  If  such 


Fig.  39.— Cut  potatoes  showing  the  different  types  of  decomposition  (f  nat.  size). 

Upper  row:  First  appearance  of  brown  spots.  Second  row:  Change  to  dry  rot. 

Third  row:  Change  to  bacterial  rot.  Lower  row:  Purely  bacterial  decomposition. 

material  is  kept  for  some  time  exposed  to  the  air,  so-called  dry  rot  sets  in, 
that  is,  the  tissue  becomes  a dry,  powdery  mass  without  the  direct  par- 
ticipation of  bacteria  ; but  bacteria  become  active  whenever  such  potatoes 
are  kept  in  an  atmosphere  that  is  saturated  with  humidity.  Under  these 
conditions  anaerobic  but yric  acid  bacteria  become  active ; the  pectic  sub- 
stances and  part  of  the  starch  are  decomposed,  and  a soft,  ill-smelling  ma- 
terial fills  the  skin.  If  the  air  was  entirely  excluded,  not  only  the  cells 
but  the  enzymes,  too,  are  killed ; the  brownish  discoloration  is  lacking  in 
this  case  and  the  butyric  acid  bacteria  alone  are  active.  Figure  39  illus- 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  FOODSTUFFS  159 


trates  these  various  possibilities.  The  only  way  to  avoid  these  alterations 
is  by  proper  storage  which  prevents  the  death  of  the  tissue. 

Activities  of  Bacteria  in  the  Digestive  Tract. — As  was  discussed  on 
p.  63,  multiplication,  decrease,  and  renewed  multiplication  of  the  micro- 
organisms introduced  with  the  food  takes  place  in  the  different  sections 
of  the  digestive  tract,  and  certain  changes  in  the  composition  of  the  micro- 
flora occur  in  accordance  with  the  changes  of  environment.  The  role 
played  by  this  intestinal  flora  has  been  extensively  investigated.  After 
many  experimental  difficulties  had  been  overcome,  it  was  repeatedly  as- 
certained that  it  is  possible  to  raise  lower  and  higher  animals  under  per- 
fectly sterile  conditions.1  The  presence  of  an  intestinal  microflora  is 
therefore  not  absolutely  necessary,  but  in  different  respects  it  is  decidedly 
useful. 

In  the  first  place,  bacteria  may  participate  in  the  digestive  processes. 
It  is  true  that  the  animal  organism  produces  numerous  digestive  enzymes 
and  that  they  are  of  primary  importance,  but  none  of  them  seems  to  be 
able  to  attack  the  cellulose,  which  constitutes  a relatively  large  percentage 
in  the  animal  diet,  especially  in  that  of  the  ruminants.  The  activity  of 
cellulose  dissolving  bacteria  is  of  fundamental  importance  in  this  case.  It 
is  mainly  due  to  their  cooperation  that  the  ruminants  can  make  use  of  such 
large  amounts  of  grass,  hay,  and  straw,  which  ability  makes  them  of  such 
great  economic  importance.  Fui'thermore,  bacteria  support  and  in- 
crease the  effect  of  the  digestive  enzymes  of  the  animal  body  in  several 
directions,  although  these  activities  are  less  essential  than  is  the  dissolu 
tion  of  the  cellulose. 

In  the  second  place,  the  products  of  the  metabolism  of  the  intestinal 
bacteria  may  exert  a beneficial,  but  sometimes  a detrimental  influence 
upon  the  digestion,  and  thereby  upon  the  general  functioning  of  the 
higher  organism.  Again  the  activity  of  acid  producing  bacteria  is  of 
special  advantage,  because  an  acid  reaction  checks  putrefactive  processes, 
which  otherwise  may  lead  to  the  appearance  of  more  or  less  toxic  sub- 
stances. 

In  the  third  place,  the  presence  of  the  intestinal  bacteria  and  of  their 
metabolic  products  is  decidedly  useful  whenever  pathogenic  bacteria  may 
enter  the  animal  body  with  the  food.  A few  pathogenic  microorganisms 
are  soon  overgrown  by  the  many  millions  of  intestinal  bacteria,  and  the 
adaptation  of  the  body  to  the  various  metabolic  products  of  its  intestinal 
flora  causes  a more  or  less  marked  immunity  against  bacterial  toxins. 
This  resistance  was  found  to  be  entirely  lacking  in  animals  free  of  bac- 
teria. 

1 E.  Kuster,  Arb.  Kaiserl.  Gesundh.  Amt.,  vol.  48,  1914,  p.  1;  E.  Wollman,  Bull. 
Inst.  Pasteur,  vol.  12,  1914,  p.  921. 


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Intestinal  and  Fecal  Microflora. — In  accordance  with  the  conditions 
offered  to  the  bacteria  in  the  different  parts  of  the  digestive  tract,  a 
natural  selection  takes  place  among  the  species  continually  introduced 
with  the  food.  Many  of  them  succumb,  while  others  are  favored 
by  their  new  environment  and  multiply  rapidly.  The  result  is  that  the 
latter  form  a rather  constant  intestinal  flora,  while  the  former  represent 
a more  accidental  occurrence.  Regularly  present  in  large  numbers  are 
the  intestinal  lactic  acid  bacteria,  that  is,  the  motile  B.  coli  and  the  im- 
motile  B.  aerogenes,  while  the  lactic  acid  streptococci  and  the  lactobacilli 
are  usually  not  very  numerous,  except  when  milk  is  fed,  as  to  calves. 
Butyric  acid  bacilli  and  other  anaerobic  and  aerobic  sporulating  species 
are  very  common,  as  are  B.  fluorescens,  proteus,  and  related  organisms. 

The  food  consumed  influences  the  intestinal  and  fecal  microflora  not 
so  much  by  its  own  microflora  as  by  its  chemical  qualities,  which  lead  to 
more  or  less  far-reaching  changes  in  the  composition  of  the  intestinal 
flora,  and  may  induce  the  development  of  varietal  characters  which  often 
exert  a very  marked  effect  upon  the  milk  and  dairy  products,  if  these  are 
subject  to  fecal  contaminations.  For  instance,  if  lactobacilli,  such  as 
are  present  in  the  intestine  of  milk  fed  animals,  are  added  to  other  food, 
very  few  of  them  will  survive  in  the  digestive  tract,  but  if  milk  or  lactose 
is  fed  persistently,  they  will  reappear  and  multiply  enormously. 


CHAPTER  IX 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 

Milk  and  dairy  products  are  of  exceptional  importance  for  human 
nutrition,  and  the  monetary  value  of  the  quantities  annually  produced  is 
very  large.  As  is  well  known,  bacterial  growth  and  activity  in  milk  may 
cause  serious  losses,  and  it  was  due  to  this  fact  that  investigations  upon 
the  abnormal  alterations  of  milk  were  part  of  the  earliest  work  done  in 
bacteriology  (see  pp.  8 and  89).  Theoretically  it  would  be  best  to 
protect  milk  from  all  contaminations,  and  to  supply  the  consumer  with 
a sterile  product.  Practically,  however,  the  necessity  of  keeping  the  cost 
of  milk  production  as  low  as  possible,  is  usually  the  first  item  to  be  com 
sidered.  The  desire  of  the  consumer  to  get  milk  at  the  lowest  possible 
price,  collides  seriously  with  the  request  of  hygienists  to  have  the  milk 
supplied  free  from  undesirable  microorganisms.  To  reconcile  both  points 
of  view  as  far  as  possible,  the  milk  producer  needs  an  adequate  knowledge 
of  all  pertinent  facts,  which  have  been  thoroughly  established  during 
the  last  decades  by  dairy  bacteriologists  in  all  parts  of  the  world. 

1.  GERM  CONTENT  OF  MILK 

The  chances  for  contamination  to  which  milk  is  exposed  on  its  way 
from  the  cow’s  udder  to  the  household,  are,  of  course,  very  numerous. 
They  begin  in  the  udder  itself  and  are  dependent  on  the  manner  in 
which  the  milking  is  done  and  delivery  to  the  consumer  is  made.  Ac- 
cording to  circumstances,  the  various  possibilities  of  contamination  are 
to  be  investigated,  and  to  be  eliminated  if  this  is  feasible. 

Bacteria  Within,  the  Udder. — As  was  mentioned  before  (p.  64),  the 
cells  of  a healthy  udder  are  free  of  bacteria,  as  is  the  blood,  and  the 
milk  is  sterile  at  the  moment  when  it  is  formed.  However,  as  soon  as  it 
passes  down  the  ducts  of  the  udder  and  accumulates  within  the  cistern, 
contamination  usually  takes  place.  Occasionally,  young  healthy  animals 
are  found  from  which  small  samples  of  milk  can  be  obtained  free  of  all 
germs,  but  these  are  rather  rare  exceptions.  As  a rule,  about  200  to  500 
bacteria  per  ec.  have  been  counted  in  milk  from  healthy  cows  when  the 
milking  was  done  aseptically  and  the  vessels  used  had  been  sterilized 
before. 


161 


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Only  a few  species  of  bacteria  are  regularly  present  in  healthy 
udders,  viz.  certain  strains  of  micrococci  and  of  streptococci.  A fat 
splitting  variety  of  B.  abortus  seems  also  to  be  rather  frequent,1  although 
not  so  constant  as  the  other  two  kinds.  Other  species,  especially  some 
causing  abnormal  alterations  in  milk,  may  appear  temporarily,  but  they 
never  become  permanent  inhabitants  of  the  healthy  cow’s  udder.  The 
reason  for  this  is  that  the  healthy  animal  tissue  possesses  to  a certain  de- 
gree similar  bactericidal  properties  as  are  characteristic  of  the  healthy 
blood.  Strictly  non-pathogenic  bacteria  may  invade  the  udder  through 
tiie  teats,  but  they  are  quickly  suppressed  and  killed.  The  micrococci 
and  streptococci  first  mentioned  are  also  avirulent,  as  is  the  fat  splitting 
variety  of  B.  abortus,  but  all  three  are  closely  related  to  pathogenic 
forms  ( Microc . pyogenes,  Streptoc.  pyogenes,  and  B.  abortus,  the  causa- 
tive agent  of  infectious  abortion  of  cattle),  and  therefore  able  to  over- 
come to  some  extent  the  resistance  offered  by  the  healthy  tissue.2  It  is 
a very  peculiar  adjustment  and  an  accurately  balanced  equilibrium  be- 
tween these  microorganisms  and  the  animal  body ; they  are  not  killed,  but 
their  multiplication  is  restricted,  at  least  as  long  as  the  animal  resistance 
is  not  weakened.  If  this  occurs,  however,  as  in  cold  rainy  weather,  or 
in  sultry  summer  heat,  or  by  blows  against  the  udder,  the  chances 
are  immediately  offered  for  these  bacteria  to  overcome  the  animal  resis- 
tance, to  multiply  rapidly,  to  regain  their  suppressed  virulence,  and  to 
cause  what  is  commonly  known  as  spontaneous  inflammation  (mastitis) 
of  the  udder. 

These  facts  are  of  very  great  importance  to  the  milk  producer,  as  well 
as  to  the  milk  hygienist.  Only  when  micrococci  and  streptococci  are 
present  in  large  numbers  in  the  milk,  is  the  suspicion  justified  that 
inflammation  of  the  udder  exists,  is  developing,  or  has  been  passed.  If 
certified  milk  is  being  produced,  such  abnormal  milk  must  be  excluded, 
although  it  may  be  still  serviceable  for  other  purposes.  If  mastitis  has 
fully  developed,  and  especially  if  pus  and  blood  are  present,  no  further 
use  of  the  milk  is  permissible.  In  regard  to  the  udder  variety  of  B. 
abortus,  thus  far  no  had  effects  have  been  recorded  in  America,  but  in 
Mediterranean  countries  a disease  called  Malta  fever,  is  widely  spread 
by  goat  ’s  milk  that  harbors  a closely  related  organism.3 

If  pathogenic  organisms  circulate  in  the  cow’s  blood,  it  is  easily  pos- 
sible that,  they  will  appear  in  the  milk,  as  is  not  infrequent  with  B. 

*11.  C.  Cooledge,  Michigan  Agr.  Exp.  Stat.  Tech.  Bull.  33,  1916,  and  41,  191S; 
A.  C.  Evans,  Jour.  Infect.  Diseases,  vol.  18,  1916,  p.  437;  vol.  23,  1918,  p.  354;  Jour. 
Bad.,  vol.  2,  1917,  p.  185. 

2 W.  Steck,  Landw.  Jahrb.  d.  Schweiz,  1921,  pp.  511-629. 

3 Z.  Ivhaled,  Jour.  Hyg.,  vol.  20,  1921,  p.  319. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK  , 163 


tuberculosis.  It  is  equally  possible  that  some  of  the  pathogenic  strep- 
tococci and  micrococci  may  enter  the  udder  with  the  blood.  But  in  most 
cases  the  invasion  comes  from  the  outside,  because  the  udder  is  always 
exposed  to  heavy  contaminations  from  dust  and  feces.  The  small  open- 
ing of  the  teat  is  wide  enough  to  admit  all  bacteria,  and  the  milk  droplets 
often  remaining  there  after  milking  permit  a rapid  multiplication  of  the 
invaders,  which  later  advance  upward  into  the  cistern  and  the  ducts 
of  the  udder.  B.  coli,  the  common  fecal  organism,  is  not  as  fre- 
quent in  the  udder  as  might  be  expected,  but  occasionally  it  appears  in 
large  numbers  and  may  become  the  cause  of  another  form  of  mastitis. 
This  is  also  true  in  regard  to  Bad.  pyocyaneum. 

Fecal  Contaminations. — The  very  high  germ  content  of  feces  was 
pointed  out  before  (p.  63),  and  it  goes  without  saying  that  clean  milk 
can  not  be  obtained  from  unclean  animals.  Millions  of  bacteria  are  car- 
ried into  the  milk  by  every  dirt  particle  loosened  from  the  cow’s  udder 
or  other  parts  of  her  body,  and  it  is  well  worth  knowing  that  even  the 
inner  parts  of  the  teats  of  dirty  udders  may  be  so  heavily  infested  with 
all  kinds  of  bacteria  that  it  will  take  weeks  after  the  cow  is  thoroughly 
cleaned  and  groomed,  before  milk  of  low  germ  content  is  obtained.  That 
the  first  few  cubic  centimeters  of  such  milk  must  be  very  rich  in  germs 
needs  no  special  explanation.  The  following  examples1  illustrate  the 
general  situation: 


Bacteria  per  cc.  Milk 

First  Part 

Middle 

End  of  Milking 

From  very  clean  animals 

600 

40 

10 

From  moderately  clean  animals . 

55,000-97,000 

2,000-10,000 

0-500 

From  dirty  animals 

6,500,000-86,000,000 

? 

12,000-43,000 

If  the  udder  is  very  dirty  it  should  be  washed  with  tepid  water  and 
soap,  wiped  dry  with  a soft  towel,  and  the  teats  greased  with  some 
neutral  fat  (vaselin).  If  it  is  fairly  clean,  wiping  with  a dry  soft  cloth 
and  light  greasing  is  sufficient.  Of  course,  no  more  vaselin  should  be 
used  than  is  necessary  to  make  the  surface  smooth  and  to  fix  the 
bacteria  left  firmly  to  the  skin.2 

Influence  of  Milking. — The  persons  who  do  the  milking  or  are  other- 
wise handling  milk  should  be  clean  and  healthy.  Carriers  of  disease 
(see  p.  140),  especially  those  of  typhoid  and  diphtheria,  should  never  be 

1 More  references  are  given  in  Chapter  III,  1,  of  the  “Handbuch  der  landwirtschaft- 
Iichen  Bakteriologie.” 

2 K.  Volmer,  “ Ueber  die  beste  Keimfreimachung  des  Euters.”  Diss.  Bern.,  1909, 
abstr.  Milchw,  Zentralbl-,  vpl,  7,  p.  175. 


164 


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allowed  in  such  places.  That  from  the  dirty  hands  of  a milker  many 
organisms  may  enter  the  milk  is  self-evident,  especially  if  the  hands  are 
kept  wet  while  milking.  If  a man  is  not  accustomed  to  milking  with  dry 
hands,  he  should  grease  them  lightly,  after  they  have  been  washed  and 
dried.  The  milker’s  garment  should  be  equally  clean.  Large  washable 
coats  or  aprons  are  best;  they  may  be  sterilized,  if  certified  milk  is 
produced.  It  is  to  be  highly  recommended  that  the  hands  be  washed 
again  before  another  cow  is  milked,  otherwise  mastitis  streptococci  may 
be  carried  from  diseased  to  healthy  animals.  The  first  milk,  which  is 
richest  in  germs  and  of  low  fat  content,  should  be  milked  into  a separate 
small  container  and  discarded.  It  should  not  be  milked  on  the  ground, 
as  is  often  done,  because  in  this  way  mastitis  bacteria  find  another  oppor- 
tunity of  being  carried  from  cow  to  cow. 

That  an  unskilled  and  careless  milker  will  cause  greater  contamina- 
tion of  the  milk  than  a skilled  and  careful  one,  is  beyond  doubt ; actual 
tests  have  shown  that  the  difference  may  be  ten-fold  and  more.  Covered 
pails  of  simple  construction,  so  that  all  parts  of  them  are  easily  accessible 
to  thorough  cleaning,  are  far  superior  to  open  pails.  The  cover  keeps  many 
dirt  and  dust  particles,  hair,  dandruff,  etc.  from  falling  into  the  milk. 
Accordingly,  the  germ  content  of  milk  in  covered  pails  is  usually  found 
to  be  only  1/5  as  high  as  that  in  open  vessels. 

Milking  machines,  unless  constructed  with  care  and  kept  scrupulously 
clean,  increase  the  germ  content  of  milk.  Even  comparatively  minor 
details,  such  as  imperfect  check  valves,  may  be  the  cause  of  heavy  con- 
taminations.1 Thorough  cleaning  is  insufficient,  unless  followed  by  a 
more  or  less  complete  sterilization  of  the  whole  apparatus.  If  a chemical 
treatment  is  considered,  a combination  of  brine  and  hypochlorite  has  been 
found  most  satisfactory,2  but  very  promising  results  have  also  been  ob- 
tained by  heating  the  carefully  cleaned  apparatus  immersed  in  water  in 
a covered  boiler  for  15  to  30  minutes  at  75°  to  85°  C.,  and  leaving  it  in 
the  covered  container,  protected  against  all  contaminations,  until  it  is  to 
be  used  again.3  Theoretically,  the  latter  procedure  is  superior,  but  not 
all  rubber  stands  the  heat;  in  such  cases  the  chemical  treatment  must 
be  relied  upon.4 

Influence  of  Utensils. — From  every  container  and  every  apparatus 
with  which  the  milk  comes  into  contact,  from  the  milking  pail  to  the  de- 

1 R.  S.  Breed  and  J.  W.  Bright,  New  York  State  Agr.  Exp.  Stat.  Bull.  4SS,  1921. 

2 G.  L.  A.  Ruehle,  R.  S.  Breed  and  G.  A.  Smith,  New  York  State  Agr.  Exp.  Stat. 
Bull.  450,  1918. 

3 G.  H.  Hart  and  W.  H.  Stabler,  Jour.  Dairy  Science,  vol.  3,  1920,  pp.  33-51. 

4 R.  S.  Breed,  Jour.  Dairy  Science,  vol.  5,  p.  102,  1922;  A.  H.  Robertson,  M.  W. 
Finch  and  R.  S.  Breed,  New  York  State  Agr.  Exp.  Stat.  Bull.  492,  1922. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


165 


livery  bottle,  a few  or  many  bacteria  are  carried  along,  continually 
swelling  the  total  germ  content.  The  following  figures  may  serve  as  an 
illustration : 1 


Germ  Content  of  Milk  Per  cc. 

In  the  milking  pail 19,000 

In  a second  container 28,000 

After  passage  of  cooler 38,000 

In  a third  container 78,000 

After  bottling 102,000 


Cleanliness  alone  is  again  not  a sufficient  precaution  if  a low  germ 
content  is  desired.  Especially  if  cleansing  soda  is  freely  applied,  it  may 
happen  that  first  of  all  the  lactic  acid  bacteria  are  suppressed,  while 
other  species  prove  more  resistant;  the  milk  produced  is  then  exposed 
to  various  unwelcome  alterations,  which  are  normally  checked  by  the 
lactic  acid  fermentation.  The  cleaned  and  rinsed  vessels  should  at  least 
be  quickly  dried,  so  that  the  surviving  bacteria  can  not  multiply  in  the 
remaining  droplets  of  water.  This  point  is  also  of  importance  if  the 
containers  are  steamed,  because  again  not  all  bacteria  and  hardly  any 
of  their  spores  are  killed,  and  these  together  with  new  contaminations 
may  give  rise  to  a very  numerous  and  undesirable  microflora.  It  has 
been  ascertained  repeatedly  that  such  apparently  clean,  but  in  fact  highly 
contaminated,  containers  may  raise  the  germ  content  of  the  milk  a 
hundred-  or  thousand-fold. 

Thorough  sterilization  of  pails,  cans,  and  bottles  is  assured  only  if 
they  are  placed  in  a hot  air  oven,  heated  for  a minute  to  160°  C.  or  for 
about  5 minutes  to  140°  C.,  and  then  kept  in  the  closed  apparatus  until 
they  are  used.  This  system  has  been  adopted  by  European  milk  pro- 
ducers during  the  last  twenty  years ; recently  it  has  also  been  recom- 
mended by  American  authors.2 

Unfortunately,  the  water  used  for  cleaning  it  not  always  as  pure  as  it 
should  be.  Spores  of  butyric  acid  bacteria  are  by  no  means  rare,  Bad. 
lactis  viscosum,  which  makes  milk  slimy,  is  rather  common  in  impure 
water,  and  sometimes  pathogenic  bacteria  may  occur.  Water  of  doubt- 
ful quality  should  be  tested  by  adding  some  of  it  to  clean  milk ; after  one 
or  a few  days  this  milk  is  to  be  compared  with  another  sample  of  the  same 
milk  to  which  no  water  had  been  added.  If  any  suspicion  arises  as 
to  the  presence  of  pathogenic  germs,  thorough  chlorination  of  the  water 
is  to  be  recommended. 

Influence  of  Air  and  of  Feeding. — As  was  pointed  out  above,  cov- 

1 Backhaus  und  Cronheim,  Ber.  d.  landw.  Instituts  Konigsberg,  vol.  2,  1898,  p.  17. 

2S.  H.  Ayers  and  C.  S.  Mudge,  Jour.  Dairy  Science,  vol.  4,  1921,  p.  79. 


166 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


ered  milk  pails  are  far  superior  to  open  ones,  because  they  give  to  the 
milk  sufficient  protection  against  the  “rain”  of  bacteria-laden  dust,  con- 
tinually falling  in  the  stable.  That  the  milk  should  be  transferred  at 
once  into  another  room  where  the  air  is  not  so  infected,  need  hardly  be 
emphasized,  yet  it  is  not  always  done.  The  high  germ  content  of  stable 
air  can  be  easily  demonstrated  by  exposing  Petri  dishes  containing  sterile 
gelatin  for  one  minute,  and  by  comparing  the  number  of  colonies  grow- 
ing on  such  plates  with  those  obtained  under  analogous  conditions  in 
other  rooms  or  in  the  open  air.1  If  the  litter  is  renewed,  the  manure 
removed,  or  dusty  roughage  is  fed,  the  contamination  of  the  air  is 
much  increased.  Such  manipulations  should  follow  the  milking,  or  they 
should  be  finished  at  least  one  hour  before  milking  time  so  that  the  ma- 
jority of  bacteria  and  molds  will  have  time  to  settle. 

Contamination  of  the  milk  by  the  microflora  of  food,  and  water  may 
occur  in  three  ways.  Droplets  of  water  and  particles  of  food  may  be 
thrown  into  the  air,  or  microorganisms  from  food  and  water  may  be 
transferred  to  the  feces,  or  bacteria  taken  up  through  the  mouth  may 
enter  the  blood  stream  and  get  into  the  udder;  but  this  last  named  possi- 
bility is  restricted  to  pathogenic  organisms.  If  the  food  acts  un- 
favorably upon  the  digestion,  fecal  contaminations  are  unavoidable.  If 
such  material  can  not  be  entirely  discarded,  it  should  be  mixed  with  other 
food  that  will  eliminate  its  bad  effect. 

Most  undesirable  contaminations  are  caused  by  flies.  A single  fly 
may  carry  hundreds  of  millions  of  bacteria,  and  because  these  are  mostly 
of  fecal  origin,  and  possibly  of  pathogenic  character,  a vigorous  cam- 
paign against  flies  should  be  waged  continually,  wffierever  milk  is  exposed 
to  the  air. 

Clarification  of  Milk. — Because  of  the  facts  discussed  on  pp.  40  and 
64  it  is  impossible  to  attain  a marked  reduction  of  the  germ  content  in  milk 
by  filtration  or  by  the  use  of  centrifugal  power.  Large  clumps  of  bacteria 
and  those  adhering  to  dirt  particles  can  be  removed,  but  the  majority 
will  remain  afloat.  Many  cell  compounds  are  broken  at  the  same  time, 
and  plate  counts  made  before  and  after  the  milk  has  passed  the  filter 
or  the  centrifuge  will,  therefore,  often  show  higher  numbers  in  the 
clarified  milk. 

The  desirability  of  removing  dirt,  hairs,  dandruff,  and  other  foreign 
material  from  milk  necessitates,  as  a rule,  one  or  the  other  method  of 
clarification,  which  will  be  the  more  effective  the  earlier  it  is  applied. 
Among  the  various  filters  those  with  a horizontal  arrangement  of  cloth 

'Photographs  of  such  plates  are  given  in  F.  Lohnis,  “Laboratory  Methods  in 
Agricultural  Bacteriology,’’  Plate  I. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


167 


or  cotton  are  least  desirable,  because  the  dirt  first  deposited  is  washed 
and  partially  dissolved  by  the  milk  that  follows.  Only  when  a construction 
is  chosen  that  forces  the  milk  upward  through  the  filter,  is  the  horizontal 
position  of  the  filtering  surface  not  objectionable.  Generally,  however, 
funnel  shaped  filters  are  superior,  because  here  the  dirt  can  settle  at  the 
lower  end  without  being  further  disturbed.  Cotton  is  superior  to  filter 
cloth ; if  the  latter  is  only  washed,  but  not  sterilized,  it  may  become  a 
source  of  heavy  contaminations.  A sample  of  milk  poured  through  such 
material  contained  for  example  per  cc. : 

Before  Filtration  After  Filtration 

20-420  140.000 

Changes  of  Germ  Content. — In  view  of  the  high  nutritive  value  of 
milk  it  is  to  be  expected  that  the  bacteria  which  found  their  way  into  it, 
will  multiply  rapidly,  especially  if  the  milk  is  not  cooled  immediately 
after  milking.  If  the  initial  germ  content  is  high,  it  increases,  indeed, 
very  rapidly,  but  with  clean  milk  the  case  is  different,  as  many  countings 
have  shown.  For  instance,  the  following  numbers  were  found  per  cc. 
milk : 


Fresh  After  3 Hours  After  6 Hours  After  9 Hours 

3090  920  1090  1160 

It  usually  took  18  to  24  hours  before  the  original  germ  content  was  re- 
stored, if  this  was  low  at  the  beginning. 

This  temporary  reduction  of  the  total  number  of  bacteria  present  in 
milk  is  usually  ascribed  to  bactericidal  properties  of  fresh  milk.  Dis- 
tinctly bactericidal  actions  are,  in  fact,  clearly  noticeable  with  colostrum 
milk,  if  this  is  kept  at  37°  C.  Leucocytes  (phagocytes)  as  well  as  the  milk 
serum  prove  active  in  a similar,  though  not  equally  pronounced,  manner 
to  that  characteristic  of  the  blood.  However,  only  part  of  the  bacteria 
are  really  killed,  while  others  are  merely  agglutinated  and  the  number 
of  their  colonies  growing  on  the  plates  therefore  reduced.  There  are 
still  other  species  which  are  not  influenced  at  all ; for  instance,  lactic  acid 
streptococci  begin  their  multiplication,  as  a rule,  at  once.  If  milk  is 
heated  for  20  to  30  minutes  at  60°  to  70°  C.  all  bactericidal  properties 
are  destroyed,  but  reductions  in  germ  content  may  still  be  observed.  Not 
all  bacteria  find  milk  a suitable  substrate,  and  they  will  die,  of 
course,  after  a while,  irrespective  of  any  bactericidal  action  of  the  milk. 

Influence  of  Keeping  Milk  at  Different  Temperatures. — Generally 
the  multiplication  of  the  bacteria,  that  always  follows  the  temporary  re- 
duction, will  be  the  more  delayed  the  more  the  temperature  of  the  milk 


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is  reduced  by  cooling.  For  instance,  milk  with  an  original  content  of 
5000  per  cc.  showed  the  following  numbers  per  cc.  after  24  hours : 

At  5°  C.  At  10°  C.  At  18°  C.  At  35°  C. 

2400  7000  280,000  12,500,000 

Because  lactic  acid  bacteria  multiply  very  little  at  or  below  10°  C., 
this  temperature  (50°  F.)  is  usually  adopted  as  sufficiently  low  and  yet 
not  too  difficult  to  reach  and  to  maintain  under  practical  conditions.  It 
would  be  much  more  expensive  to  lower  the  temperature  to  5°  C.  or 
less,  and  the  effect  would  not  justify  the  increase  in  costs,  because  be- 
tween 0°  and  5°  C.  different  psychrophilic  bacteria  will  multiply,  among 
them  B.  fluorescens  and  certain  micrococci,  which  attack  casein  and  fat 
and  cause  a distinct  deterioration  of  taste  and  flavor.  The  following 
counts  were  obtained  per  cc.,  when  samples  of  fresh  and  of  pasteurized 
milk  were  kept  at  4°  to  5°  C. : 


Counts  per  cc. 

1 Day 

2 Days 

3 Days 

4 Days 

5 Days 

6 Days 

Fresh  milk 

Pasteurized  milk 

21,120 

60 

23,680 

40 

121,080 

30 

338,560 

360 

innumerable 
32,040  209,920 

Curves  showing  the  multiplication  of  some  of  the  most  common  milk 
bacteria  (Streptoc.  laetis,  B.  coli,  and  fluorescens),  when  grown  sepa- 
rately and  in  symbiosis  at  different  temperatures,  were  presented  in 
Fig.  17  (p.  57). 

Influence  of  Transporting  and  Marketing  Milk. — It  is  not  too  diffi- 
cult to  produce  milk  of  low  germ  content,  if  all  cows  are  healthy  and, 
so  far  as  practically  possible,  all  sources  of  contamination  are  care- 
fully avoided.  5000  to  10,000  bacteria  per  cc.  of  certified  milk,  and 
50,000  to  100,000  per  cc.  of  ordinary  milk  can  be  accepted  as  maximum 
at  the  places  of  milk  production,  but  when  such  milk  is  tested  at  its  place 
of  destination,  often  one  or  several  million  bacteria  per  cc.  are  found, 
due  to  improper  handling  in  transit.  Use  of  unclean  vessels  for  trans- 
portation and  lack  of  protection  against  high  temperature  of  the  air  are 
the  two  main  factors  which  frequently  nullify  all  efforts  of  careful  milk 
producers.  If  the  transport  vessels  are  sent  back  to  the  producer  with- 
out being  thoroughly  cleaned  and  sterilized,  all  kinds  of  undesirable  bac- 
teria have  excellent  opportunities  of  rapid  multiplication,  and  every 
cubic  centimeter  of  clean  milk  filled  into  such  vessels  is  exposed  to  very 
serious  contaminations. 

In  the  second  place,  insufficient  protection  against  high  outside  tem- 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK  169 


perature  may  accelerate  the  multiplication  of  the  milk  bacteria  to  such 
an  extent  that  many  millions  per  cc.  may  be  present  when  the  milk 
reaches  its  destination.  General  use  of  refrigerator  cars  for  the  trans- 
portation of  milk  is  very  desirable,  and  the  stipulation  contained  in  milk 
ordinances  that  milk  should  always  be  kept  at  a temperature  not  higher 
than  50°  F.  is  very  appropriate  and  useful,  if  properly  enforced. 

Carelessness  in  handling  the  milk  in  the  household  frequently  adds 
to  the  bad  effect  of  long  unprotected  transportation  in  hot  weather,  and 
the  lower  the  original  germ  content  has  been,  the  larger  will  be  its  rela- 
tive increase  under  such  unsuitable  conditions.  The  number  of  bacteria 
present  at  the  time  of  delivery  will  increase  about  100-  to  200-fold  when 
the  milk  is  kept  from  morning  until  evening  at  summer  temperature  out- 
side of  the  refrigerator.  Compared  with  this  last  accumulative  effect,  all 
previous  steps  are  of  comparatively  little  importance.  Certified  milk 
with  originally  5000  bacteria  per  cc.  may  ultimately  harbor  not  less  than 
2,000,000,  and  ordinary  milk  which  left  the  farm  with  a germ  content  of 
100,000  per  cc.,  will  show  not  less  than  10  millions,  if  handled  in  this 
faulty  manner. 

Germ  Content  of  Different  Kinds  of  Milk. — The  establishment  of 
three  classes  of  milk — certified  milk  with  less  than  10,000  bacteria  per  cc., 
Grade  A milk  with  less  than  100,000  bacteria  per  cc.,  and  pasteurized 
milk — is  probably  the  relatively  best  arrangement  which  can  be  made. 
In  several  parts  of  the  United  States  such  or  similar  regulations  are 
in  force,  while  in  other  districts,  and  especially  in  European  countries, 
many  different  arrangements  have  been  adopted.  It  is,  of  course, 
next  to  impossible  to  harmonize  the  points  of  view  held  by  milk 
producer,  milk  dealer,  milk  consumer,  milk  hygienist,  and  milk  chemist. 
Raw  milk  of  very  low  germ  content  and  absolutely  free  of  all  pathogenic 
or  otherwise  harmful  bacteria,  that  is,  certified  milk,  is  needed  only  in 
those  cases  where  children  do  not  thrive  on  pasteurized  milk.  The  costs  of 
production  and  handling  are  necessarily  high,  but  if  they  save  a child's 
life,  they  are  well  spent.1  Milk  of  moderate  germ  content,  that  is  below 
100,000  or  200,000  per  cc.,  represents  an  excellent  talfile  milk,  provided 
that  care  is  taken  to  exclude  pathogenic  bacteria,  as  well  as  milk  of 
abnormal  taste  and  flavor.  Its  production  is  less  costly  than  that  of 
certified  milk,  but  sterilization  of  the  utensils  and  careful  cooling  of  the 
milk  are  necessary  also  in  this  case,  and  must  be  paid  for  by  the  con- 
sumer. 

The  higher  germ  content  of  all  other  milk  may  be  reduced  by  pas- 
teurization, which  is  relatively  the  cheapest  method  of  rendering  infected 
1 For  details  in  regard  to  production  and  use  of  certified  milk  see  Proceedings  of  the 
Annual  Conferences  of  the  American  Association  of  Medical  Milk  Commissions. 


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milk  harmless.  However,  one  point  is  to  be  strongly  emphasized  concern- 
ing the  general  use  of  pasteurized  milk.  It  is  not  very  difficult  to  kill 
practically  all  bacteria  that  have  grown  in  the  milk,  but  their  bodies,  as 
well  as  their  metabolic  products,  are  not  removed,  and  if  the  milk  is  not 
quickly  cooled  and  always  kept  below  50°  F.  after  pasteurization,  the 
few  surviving  bacteria  will  multiply  very  rapidly  and  may  cause  altera- 
tions in  the  milk  that  will  make  it  a dangerous  food,  especially  for 
little  children. 

Simple  determinations  of  the  germ  content  of  milk  are  of  re- 
stricted value,  and  the  results  obtained  in  this  way  should  not  be  over- 
estimated. This  holds  true  especially  insofar  as  the  scoring  of  dairy 
farms  and  dairies  is  concerned.  Some  authors  are  inclined  to  use  the 
bacteria  counts  as  a means  to  prove  the  inaccuracies  of  the  various  scoring 
systems.1  However,  if  both  determinations  are  properly  made  and  the 
investigations  are  placed  upon  a broad  basis,  a fairly  close  parallelism 
is  to  be  expected  and  has  been  actually  observed.2 

The  situation  is  similar  in  regard  to  the  relations  existing  between 
dirt  content  and  germ  content  of  milk.  That  there  is  no  close  parallelism 
between  the  two  findings  in  individual  cases  has  been  shown  by  European 
investigators  20  or  30  years  ago,  and  it  is  practically  self-evident.  Even 
if  milk  is  handled  in  a rather  unclean  manner  it  will  rarely  contain 
more  than  10  mg.  of  dirt,  that  is  cow’s  feces,  in  1000  cc.  According  to 
the  data  presented  on  p.  63,  approximately  150  million  of  bacteria 
would  be  brought  into  the  milk,  or  150,000  per  c.c.,  that  is  less  than  is 
often  added  by  a contaminated  container.  Furthermore,  visible  dirt 
can  be  easily  removed  by  filtration  or  centrifugation,  while  the  germ  con- 
tent of  such  milk  is  not  lowered  thereby.  If,  however,  filter  tests  are 
regularly  made  with  many  milk  samples  that  have  not  been  previously 
strained,  it  becomes  obvious  that  the  determination  of  the  dirt  content 
gives  a fairly  reliable  indication  of  the  bacterial  quality  of  these  milk 
samples,  not  only  in  regard  to  the  number,  but  also  concerning  the  kinds 
of  bacteria  present.  Next  to  the  presence  of  pathogenic  organisms  a 
heavy  fecal  contamination  is,  of  course,  least  desirable. 

Harmless  and  Pathogenic  Bacteria  in  Milk. — One  or  several  millions 
of  bacteria  occupy  very  little  space  in  the  milk,  as  was  illustrated  on  pp. 
18  and  64,  and  it  goes  without  saying  that  ordinary  milk  contain- 
ing 10  or  100  millions  of  lactic  acid  bacteria  is  far  superior  to  milk  with 
a low  total  germ  content,  but  not  absolutely  free  from  pathogenic  organ- 
isms. Tuberculosis,  typhoid,  scarlet  fever,  and  diphtheria  are  occasion- 

1 J.  A.  Harris,  Science,  vol.  42,  1915,  p.  503;  Ch.  E.  North,  Amer.  Jour.  Publ. 
Health,  vol.  7,  1917,  p.  25. 

2 1.  V.  Hiscock,  Jour.  Dairy  Science,  vol.  5,  1922,  p.  83, 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK  171 


ally  spread  by  milk,  and  if  the  udder  harbors  pathogenic  streptococci,  or 
the  cows  are  infected  with  foot  and  mouth  disease,  such  milk  may  also 
cause  diseases  in  the  human  mouth,  throat,  and  digestive  tract.  Many 
special  tests  have  been  invented  to  detect  the  presence  of  harmful  bac- 
teria in  the  milk,  but  the  results  obtained  are  not  very  satisfactory. 
Animal  tests  are  necessary,  for  instance,  to  prove  the  absence  or  presence 
of  tubercle  bacilli  in  milk,  and  it  takes  weeks  before  a conclusion  can  be 


Fig.  40. 


— 10ec 


Fig.  41. 


Fig.  40. — Centrifuge  for  testing  milk  (\  nat.  size). 
Fig.  41. — Milk  glass  for  mastitis  test  (§  nat.  size.) 


reached.  Milk-borne  infections  of  typhoid,  scarlet  fever,  and  of  diph- 
theria can  be  traced  only  by  careful  examinations  made  by  hygienists. 
This  is  the  reason  why  pasteurization  or  boiling  of  the  milk  is  to  be  recom- 
mended in  all  cases  where  the  hygienic  quality  of  the  milk  supply  is  not 
known. 

Mastitis  or  Leucocyte  Test. — The  detection  of  a contamination  by 
streptococci  from  an  inflamed  udder  is  compara- 
tively easy.  If  10  cc.  samples  of  the  milk  are 
centrifuged  in  an  apparatus  like  that  shown  in 
Pigs.  40  and  41,  and  in  the  tapering  end  of  the 
test  glass  a yellowish  precipitate  is  thrown  down 
which  comes  close  to  or  surpasses  the  lines  marked 
1 and  2,  equal  to  1 or  2 parts  per  1000,  a micro- 
scopic examination  of  the  sediment  is  to  be  made. 

If  at  1000-fold  magnification  a picture  is  obtained  Fig.  42. — Sediment  of 
similar  to  that  in  Pig.  42,  the  suspicion  is  justified  mastltls milk,  stained, 
that  the  milk  contains  secretions  from  a diseased 
udder.  Presence  of  blood  in  the  precipitate  increases  this  probability. 


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However,  no  final  decision  can  be  based  upon  this  preliminary  test.  Milk 
drawn  from  healthy  udders  may  sometimes  contain  rather  large  quanti- 
ties of  leucocytes,  which  form  a heavy  yellowish  precipitate  when  the 
milk  is  centrifuged,  and  streptococci  from  other  sources  may  grow  in  the 
milk  and  have  the  appearance  of  mastitis  streptococci.  Therefore,  the 
examination  must  be  extended  to  the  place  from  which  the  milk  was 
obtained,  and  it  must  be  ascertained  by  individual  tests  whether  some 
of  the  milk  produced  contains  leucocytes  (pus  cells)  as  well  as  numerous 
streptococci.  If  this  is  the  case,  a clinical  examination  will  almost 
invariably  show  that  the  udder  of  the  particular  animal  is  inflamed. 

Counting  Bacteria  in  Milk. — For  a long  time  plate  counts  only  -were 
made  for  determining  the  total  germ  content  of  milk,  but  more  recently 
microscopic  examinations  are  made  in  increasing  numbers.  The  latter 
are  more  reliable,  especially  if  their  results  are  compared  with  those  ob- 
tained on  thickly  sown  plates  of  beef  agar  kept  only  for  a day  or  two. 
The  necessity  to  get  the  results  as  early  as  possible,  increases  the  useful- 
ness of  microscopic  tests,  while  on  the  other  hand  their  value  is  impaired 
by  the  fact  that  accurate  counts  are  not  secured  if  the  total  germ  con- 
tent is  relatively  low,  as  in  Grade  A and  certified  milks. 

A quick  and  fairly  accurate  determination  of  the  total  germ  content 
is,  of  course,  very  desirable  especially  in  these  two  cases,  and  such  a test 
has  become  possible  by  a combination  of  plate  and  microscopic  examina- 
tion is  the  so-called  little  plate  method.1  1/10  or  1/20  cc.  of  milk  is  mixed 
with  agar,  and  the  mixture  is  spread  on  a measured  area  of  a sterilized 
slide,  which  is  kept  at  38°  C.  for  6 to  8 hours;  then  the  layer  is  dried  and 
stained,  and  the  small  colonies  are  counted  under  the  microscope.  Not 
every  cell  will  have  formed  a colony  at  this  time,  but  for  practical  pur- 
poses the  results  obtained  are  sufficiently  reliable. 

Methylene  Blue  Reduction  Test. — A most  simple  way  of  grading 
milk  on  a bacteriological  basis,  without  making  use  of  intricate  bacterio- 
logical methods,  has  become  accessible  since  it  was  discovered  that  many 
stains  when  added  to  milk  lose  their  characteristic  color  sooner  or  later 
and  are  turned  white  by  bacterial  action.  Among  the  dyes  tested  methyl- 
ene blue  has  proved  most  satisfactory.  If  a cubic  centimeter  of  a highly 
diluted  solution  of  this  dye  is  mixed  with  a small  sample  of  milk 
(10-40  cc.),  the  latter  shows  a faint  but  distinct  bluish  color  that  vanishes 
after  a few  minutes  or  after  several  hours  according  to  the  high  or  low 
germ  content  of  the  milk.  The  bacteria  are  said  to  “reduce”  the  blue 
stain  to  a white  leuco-eompound,  and  therefore  the  test  has  been  called 
reduction  test,  although  it  is  in  fact  hydrogen  and  other  substances  pro- 

1 W.  D.  Frost,  Jour.  Infect.  Diseases,  vol.  28,  1921,  p.  176. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


173 


duced  by  the  bacteria  or  present  in  the  milk  itself  that  cause  the  change.1 
Pipettes,  test  tubes,  and  a water  bath  kept  at  38°  to  40°  C.,  is  all  the 
apparatus  needed  for  this  test. 

In  the  Scandinavian  countries,  where  the  city  milk  supply  is  very  well 
organized,  the  methylene  blue  reduction  test  has  been  widely  adopted 
during  the  last  ten  years  and  is  now  generally  recognized  as  a reliable 
basis  for  grading  milk  according  to  its  germ  content,  if  the  grading  is 
done  on  a broad  basis.2  Individual  tests  are  liable  to  furnish  somewhat 


Fig.  43. — Results  of  reduction,  catalase,  and  leucocyte  tests  made  with  90  samples 
of  milk  of  different  germ  content. 


conflicting  results,  but  the  average  of  a sufficiently  large  number  of 
tests,  for  example  the  monthly  average  figure  of  each  milk  producer,  is 
generally  accepted  as  very  reliable.  It  is  indispensable,  of  course,  that 
the  concentration  of  the  stain  added  to  the  milk  is  always  the  same.  For 
this  purpose  tablets  are  manufactured  by  the  firm  Blauenfeldt  & Tvede 
in  Copenhagen,  Denmark,  which  are  used  internationally. 

The  uppermost  curve  in  Fig.  43  shows  what  results  may  be  obtained 

1 R.  Burri  und  J.  Kursteiner,  Milchw.  Zentralbl.,  vol.  41,  1912,  pp.  41,  68;  E.  B. 
Fred,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  35,  1912,  p.  391. 

2 Chr.  Barthel  und  Orla  Jensen,  Milchw.  Zentralbl.,  vol.  41,  1912,  p.  417. 


174 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


in  such  reduction  tests.  The  general  tendency  to  decline  with  rising 
bacterial  counts  is  as  conspicuous  as  is  the  rather  erratic  behavior  of  in- 
dividual cases,  especially  if  the  total  germ  content  is  low.  There  are  two 
reasons  for  these  irregularities.  First,  the  accuracy  of  the  bacteriological 
counting  methods  is  more  or  less  limited,  and  in  the  second  place,  the  dif- 
ferent kinds  of  bacteria  act,  of  course,  not  uniformly  upon  the  methylene 
blue  or  on  any  other  stain.  Certain  species  cause  a rapid  change,  others 
are  slower,  and  some  remain  entirely  inactive ; the  influence  of  variation, 
age  of  growth,  etc.  are  of  similar  importance.  This  side  of  the  problem 
has  also  been  very  thoroughly  investigated  by  European  bacteriologists, 
and  it  was  noticed  that  in  general  the  least  desirable  organisms  are 
most  active,  that  is,  not  only  the  quantity  but  also  the  quality  of  the 
bacteria  present  in  the  milk  finds  an  adequate  expression  in  the  length 
of  time  needed  for  the  reduction.  Accordingly,  the  following  classifi- 
cation has  been  adopted  for  the  routine  grading  in  Scandinavian  city 
milk  supplies : 


Time  of  reduction 

More  than 

51-2  hrs. 

2 hrs -20  min. 

Less  than 

51  hrs. 

20  min. 

Approximate  germ  content. . . 

Less  than 

1-4  million 

4-20  million 

More  than 

1 million 

20  million 

Milk  grades 

I.  Good 

II.  Medium 

III.  Poor 

IV.  Very  poor 

The  curve  in  Fig.  43  shows  that  even  individual  tests  fit  this  grouping 
fairly  well.  Perhaps  the  classification  might  be  simplified  by  drawing  the 
lines  at  V2,  2,  and  6 hours ; and  the  best  grade  of  milk  might  be  singled 
out  by  repeating  the  observation  at  the  end  of  12  hours.  Undoubtedly 
the  reduction  test  will  gain  in  importance,  the  more  the  bacteriological 
quality  of  milk  is  appreciated.1 

Catalase  Test. — Another  biological  method  for  testing  milk  is  the 
so-called  catalase  test,  which  is  based  upon  the  fact  that  peroxide  of 
hydrogen  is  split  into  water  and  oxygen  by  an  enzyme  called  catalase, 
present  in  leucocytes  and  other  cellular  elements  of  the  milk  as  well  as 
in  many  bacteria.  The  liberated  oxygen  is  measured,  and  it  is  assumed 
that  clean  milk  is  characterized  by  weak,  unclean  by  strong  catalase  ac- 
tion. The  curve  of  the  catalase  tests  in  Fig.  43  shows  that  there  is  in- 
deed a slight  rising  tendency,  indicating  the  influence  of  the  higher  bac- 
terial numbers,  but  a comparison  of  the  catalase  curve  with  the  curve 

1 See  also  E.  G.  Hastings,  Jour.  Dairy  Science,  vol.  2,  1919,  p.  293;  A.  Cunning- 
ham and  B.  A.  Thorpe,  Jour.  Ilyg.,  vol.  19,  1920,  p.  107. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK  175 


for  the  sediment  of  the  leucocyte  test,  also  given  in  Pig.  43,  makes  it  evi- 
dent that  the  effect  of  the  cell  content  of  the  milk  is  much  more  pro- 
nounced than  that  of  the  bacteria.  The  leucocyte  test,  however,  is  much 
more  easily  made  than  the  catalase  test,  and  when  combined  with  micro- 
scopic tests  it  is  much  more  valuable,  as  was  explained  before. 

Acidity  and  Alcohol  Tests. — Because  the  majority  of  the  bacteria 
present  in  normal  milk  are,  as  a rule,  acid  producers,  the  determination 
of  the  acidity  of  milk  by  titration,  or  by  measuring  the  hydrogen-ion  con- 
centration, permits  a more  or  less  accurate  estimate  of  its  total  germ 
content.  But  alkali  and  rennet  producing  bacteria  are  also  not  rare,  and 
sometimes  they  are  so  numerous  that  even  the  spontaneous  curdling  of 
the  milk  will  take  place  while  the  reaction  is  still  close  to  neutral,  and 
the  hydrogen  number  not  far  from  normal. 

Superior  to  the  acidity  test  is  the  alcohol  test.  It  consists  in  mixing 
in  a test  tube  equal  parts  of  milk  and  alcohol  of  70  or  75  per  cent 
strength,  and  in  ascertaining  whether  flocculation  of  the  casein  takes 
place.  If  this  happens,  the  milk  will  probably  coagulate  when  it  is 
heated.  Therefore,  this  test  is  of  special  value  when  milk  is  to  be 
pasteurized,  condensed,  or  evaporated.1  Several  or  many  millions  of 
bacteria  are  always  present  in  such  milk.  In  addition  to  the  acidity 
and  to  the  amount  of  rennet  produced  by  them,  the  coagulation  is 
dependent  on  the  calcium  content  of  the  milk.2 

Fermentation  Test. — Another  very  practicable  and  valuable  test, 
which  however  again  requires  at  least  12  haul’s’  time,  has  long  been 
used  in  Switzerland  for  grading  milk  in  cheese  factories.  From  every 
delivery  samples  are  filled  into  four  sterilized  (boiled)  test  tubes  or 
jars;  two  of  them  receive  a few  drops  of  rennet  solution,  while  the 
other  pair  remains  without  rennet.  The  first-named  test  (with  rennet) 
is  known  in  America  as  the  Wisconsin  curd  test.  The  samples  are  kept 
in  water  of  38°  to  40°  C.  After  12,  or  better  after  24,  hours  altera- 
tions of  the  milk  and  characteristic  curd  formations  become  visible, 
such  as  are  shown  in  Plate  X.  Furthermore,  taste  and  flavor  of  the 
milk  are  to  be  tested,  and  if  methylene  blue  was  previously  added  to  the 
milk  (without  rennet)  the  results  of  the  reduction  test  are  secured 
simultaneously.  This  combined  examination  gives  to  the  dairyman  a 
very  good  general  information  upon  number  and  quality  of  microor- 
ganisms present,  without  compelling  him  to  wait  for  the  outcome  of 
detailed  bacteriological  investigations.  If  a yellow  sediment  becomes 
visible  in  the  fermentation  test  tube,  it  may  serve  as  an  indication  of 

1 U.  S.  Dept,  of  Agr.,  Bull.  944,  1921. 

2H.  H.  Sommers  and  E.  B.  Hart,  Jour.  Biol.  Chem.,  vol.  40,  1919,  p.  1 


176 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


mastitis  milk,  analogous  to  the  precipitate  in  the  special  mastitis  test 
discussed  above. 

Very  clean  milk,  which  remains  unchanged  in  the  milk  test  for  24 
hours,  gives  frequently  poor  results  in  the  curd  test  on  account  of  the 
absence  of  lactic  acid  bacteria.  If  these  are  present  in  considerable 
numbers  a homogeneous  curd  is  obtained  without,  as  well  as  with,  rennet. 
Strong  gas  formation  is  nearly  always  due  to  fecal  contaminations. 
Fermentation  tests  made  with  samples  of  milk  which  are  taken  in 
sterilized  tubes  at  all  places  where  contaminations  possibly  occur,  will 
be  very  helpful  in  discovering  any  hidden  source  of  milk  infection  in  the 
stable  or  in  the  dairy. 

2.  ACTIVITIES  OF  BACTERIA  IN  MILK 

Not  all  alterations  which  may  take  place  in  milk  are  the  work  of 
microorganisms.  Abnormalities  in  composition,  taste,  and  flavor  are 
sometimes  already  noticeable  at  the  moment  when  the  milk  leaves  the 
udder,  due  either  to  purely  chemical  influences,  as  in  colostrum  and  in 
milk  produced  close  to  the  end  of  lactation,  or  to  bacterial  infection  of 
the  udder,  as  in  mastitis  and  tuberculosis.  If  the  freshly  drawn  milk 
appears  normal  and  the  milking  is  done  strictly  aseptically,  it  will  often 
take  weeks  before  composition,  taste  and  flavor  are  markedly  changed. 
Part  of  these  alterations  are  caused  by  genuine  milk  enzymes,  but  com- 
pared with  the  enzymatic  actions  of  the  bacteria,  present  even  in  the 
cleanest  milk,  they  are  of  no  practical  importance.  Nearly  all  the  al- 
terations which  may  develop  within  the  comparatively  short  time  until 
the  milk  is  consumed,  are  the  work  of  microorganisms.  An  exception  to 
this  rule  is,  for  example,  the  unfavorable  influence  exerted  by  direct 
sunlight  upon  milk  fat,  which  assumes  an  unpleasant  flavor  if  bottled 
milk  is  exposed  for  some  time  to  bright  sunlight. 

Normal  and  Abnormal  Alterations  of  Milk. — The  only  alteration  of 
milk  which  may  be  accepted  as  normal,  and  which  is  even  desired  in 
some  cases,  consists  in  the  formation  of  lactic  acid  and  the  coagulation 
of  the  casein.  If  ordinary  milk  is  plated  on  whey  agar  to  which  chalk 
has  been  added,  usually  a picture  like  that  shown  in  Fig.  44  is  obtained ; 
most  colonies  produce  acid  enough  to  dissolve  the  chalk  in  their  im- 
mediate neighborhood,  and  a clear  halo  appears  around  them. 

If  sour  milk  is  kept  for  a few  days  exposed  to  the  air.  at  first  Oidium 
lactis  and  other  fungi  begin  to  grow  on  the  surface.  They  destroy  part 
of  the  acid,  and  begin  to  digest  the  curd.  Later  proteolytic  bacteria 
may  become  active,  and  if  the  milk  is  kept  long  enough,  ultimately  a 


Plate  X 


1.  Milk  Fermentation  Tests 


(a)  Milk 

( b ) Gelatinous 

(c)  Cheesy 

( d ) Strong 

(e)  Very  strong 

remained 

coagulation, 

coagulation, 

gas  formation 

gas  formation 

liquid 

little  whey 

much  whey 

(B.  coli) 

(B.  amylobacter) 

2.  Curd  Tests 


(a)  Smooth, 
without 
holes 


(6)  Almost 
smooth,  but 
with  holes 


(c)  Straight 
but  with 
many  holes 


(d)  Blown 

(e)  Torn 

C Facing  page  176 ) 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK  177 


brownish  liquid  of  offensive  odor  will  result.  The  absence  of  air,  how- 
ever, prevents  this  development ; the  acid  will  not  be  destroyed  and 
no  further  change  will  take  place. 

A rather  common  abnormal  alteration  of  milk  is  its  curdling  under 
the  influence  of  rennet  producing  bacteria.  Changes  in  taste,  flavor, 
color,  and  viscosity  occur  also  from  time  to  time,  although  they  are,  as 
a rule,  less  frequent  now  than  they  were  formerly,  when  little  was 
known  about  bacterial  action,  and  milk  was  kept  in  musty  cellars. 

Formation  of  Lactic  Acid. — Normal  milk  always  shows  a slight 
initial  acidity,  due  to  its  chemical  composition.  The  streptococci  and 
micrococci  that  are  almost  constantly  present  in  the  udders,  do  not 


Fig.  44. — Agar  plate  with  colonies  of  acid  producing  bacteria  (J-  nat.  size). 


exert  any  appreciable  influence  in  this  direction,  except  in  very  rare 
cases.  The  numerous  lactic  acid  bacteria,  usually  introduced  by  un- 
sterilized vessels,  also  remain  without  effect  for  some  hours.  Their  mul- 
tiplication begins  at  once,  because  they  are  not  hindered  by  the  weak 
bactericidal  properties  of  the  milk;  but  not  before  enough  enzymes  are 
produced,  will  the  conversion  of  the  milk  sugar  into  lactic  acid  become 
noticeable.  Some  authors  have  tried  to  calculate  an  “average”  effi- 
ciency of  a single  cell  of  lactic  acid  bacteria,  but  it  suffices  to  compare 
consecutive  bacterial  counts  and  acid  determinations,  simultaneously 
made  during  one  or  several  days,  to  become  convinced  that  no  constant 
relation  exists  between  number  and  efficiency.  For  example,  the  fol- 
lowing increases  (-f-)  and  decreases  ( — ) have  been  recorded:1 


O.  Rahn,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  32,  1912,  p.  375. 


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Changes  after 

9 

12 

15  18 

21 

42 

27  hrs. 

Millions  bacteria  per  cc 

+20 

+90 

+300  |+700 

+200 

+ 100 

-150 

Acidity  (ec.N/10  NaOHper  lOOOcc.) 

+ 1 

+ 1 

+4  +22 

+20 

+5 

+2 

In  other  cases  the  disparity  between  changes  in  acidity  and  in  bacterial 
numbers  was  even  more  marked,  similar  to  that  shown  in  Fig.  37 
(p.  154).  The  enzymes  survive  the  dying  cells  and  continue  to  act;  on 
the  other  hand,  the  cells  of  lactic  acid  bacteria  may  lose  more  or  less 
completely  their  ability  of  enzyme  production,  while  they  retain  their 
full  vitality.  In  the  early  stages,  about  4/s  °f  the  lactic  acid  formed 
enters  chemical  or  physical  combinations,  especially  with  milk  casein. 
The  hydrogen-ion  concentration  increases  until  approximately  pH=4.7 
is  reached,  and  then  it  remains  at  this  point  until  the  coagulation  of 
the  casein  is  completed.1 

Behavior  of  the  Four  Groups  of  Lactic  Acid  Bacteria. — The  main 

characteristics  of  the  four  groups  of  lactic  acid  bacteria  have  been  dis- 
cussed on  p.  119,  where  it  was  also  mentioned  that  other  bacteria  may  dis- 
play similar  abilities,  which  however  are  of  no  practical  importance. 
It  is  to  be  added  that  some  yeasts  are  known  to  produce  lactic  acid, 
and  that  occasionally  they  are  active  in  ripening  cream. 

According  to  their  efficiency  in  lactic  acid  production  the  members 
of  the  four  groups  may  be  classed  as  follows.  Generally,  the  smallest 
quantities  of  acid  are  produced  by  the  micrococci;  next  in  activity 
come  the  intestinal  lactic  acid  bacteria;  stronger  and  much  purer 
is  the  lactic  acid  formation  by  the  streptococci ; and  the  largest  quan- 
tities are  usually  produced  by  the  laetobacilli,  although  a comparatively 
long  time  is  required  before  the  maximum  is  reached.  0.5  to  0.8 
per  cent  lactic  acid  is  usually  the  maximum  for  the  streptococci,  1 to  2 
and  sometimes  3 per  cent  for  laetobacilli.  Naturally,  this  general  classi- 
fication does  not  fit  every  individual  case;  exceptionally  strong  and 
weak  varieties  may  be  found  in  every  group. 

Whether  one  or  the  other  group  predominates  in  milk  or  in  dairy 
products  depends  on  the  mode  of  infection,  the  temperature,  the  pres- 
ence or  absence  of  air,  and  the  composition  of  the  substrate. 

Concerning  the  mode  of  infection  it  is  to  be  pointed  out  that  the 
micrococci  come  mostly  from  the  udder,  from  the  air,  and  from  the 
utensils ; the  intestinal  lactic  acid  bacteria,  from  feces,  from  impure  water, 
and  from  unclean  vessels ; the  streptococci,  mostly  from  containers, 

1 L.  L.  Van  Slyke  and  J.  C.  Baker,  Jour.  Biol.  Chem.,  vol.  35,  1918,  p.  147. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


179 


more  rarely  from  the  cow  (udder,  hair,  feces)  ; the  lactobacilli,  from 
silage  and  other  food,  saliva,  feces,  soil,  and  cheese.  If  the  temperature 
is  kept  below  10°  C.,  micrococci  will  predominate  after  a few  days; 
around  20°  C.  streptococci  are  very  active;  30°  to  40°  C.  are  most 
suitable  for  the  majority  of  intestinal  lactic  acid  bacteria;  and  at  45° 
to  50°  C.  hardly  anything  but  lactobacilli  will  survive.  This  general 
classification  is  again  not  without  exceptions,  but  it  is  well  worth  know- 
ing that  around  20°  and  45°  C.  the  cleanest  and  most  rapid  formation 
of  lactic  acid  is  to  be  expected,  while  below  10°  C.  and  between  30°  to 
40°  C.  many  by-products  are  formed  which  act  unfavorably  upon 
taste  and  flavor.  If  milk  cultures  of  lactic  acid  bacteria  are  kept 
continually  at  high  temperatures,  practically  all  of  them  are  killed 
after  a few  days,  while  at  0°  C.  most  cultures  will  remain  alive  for  a 
long  time,  especially  if  enough  chalk  is  added  to  neutralize  the  acid 
formed.  The  presence  of  air  favors  micrococci  and  intestinal  bacteria, 
while  the  majority  of  streptococci  and  lactobacilli  grow  best  under 
anaerobic  conditions.  Their  surface  colonies  on  agar  plates  are  therefore 
small  (see  Fig.  40),  and  milk  filled  into  deep  containers  shows  more 
rapid  acidification  than  that  kept  in  flat  vessels.  The  quality  of  milk 
is  of  influence  chemically  as  well  as  biologically.  Variations  in  the 
percentage  of  sugar,  casein,  and  calcium  make  one  milk  more  or  less 
liable  than  another  to  undergo  acidification  and  coagulation.  Further- 
more, symbiotic  and  antagonistic  effects  of  the  accompanying  microflora 
may  stimulate  or  retard  the  activities  of  the  lactic  acid  bacteria.  Accord- 
ingly, strains  of  known  character  can  not  be  expected  to  act  uniformly 
under  all  circumstances,  even  if  no  spontaneous  variation  occurs. 

Formation  of  Volatile  and  Other  Acids,  of  Alcohols,  and  of  Gases. — 
Formic,  acetic,  and  succinic  acids  are  almost  constantly  present  in  sour 
milk.  The  majority  of  micrococci  and  of  intestinal  bacteria  are  in- 
clined to  produce  these  acids  in  addition  to,  or  instead  of,  lactic  acid ; 
streptococci  and  lactobacilli  may  participate  in  in  the  formation  of 
acetic  acid,  which  is  occasionally  produced  in  relatively  large  quantities 
by  the  destruction  of  the  lactates  first  formed.1  Butyric  acid  appears 
sometimes  in  pasteurized  and  incompletely  sterilized  milk.  Members  of 
the  B.  amylobacter  group  are  so  common  in  soil,  water,  fodder,  and 
feces  that  a few  such  spores  will  always  gain  entrance,  but  the  acid 
reaction  normally  produced  by  the  lactic  acid  bacteria  prevents  their 
germination  under  ordinary  circumstances. 

The  production  of  ethyl  alcohol  in  milk  is  usually  of  little  impor- 

1 E.  B.  Fred,  W.  H.  Peterson  and  A.  Davenport,  Jour.  Biol.  Chern.,  vob  39,  1919, 
p.  347. 


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tance,  but  in  fermented  milks,  such  as  kefir  and  koumiss,  1 to  2 per 
cent  or  more  may  be  produced  by  various  yeasts.  Sporulating  Sacchar- 
omycetes  as  well  as  non-sporulating  Torulaceae  are  known  to  become 
active ; some  of  them  attack  the  milk  sugar  directly,  while  in  other  cases 
the  lactose  must  first  be  changed  by  bacterial  action  to  glucose  and 
galactose,  before  the  alcoholic  fermentation  can  take  place.  Occasion- 
ally some  bxityl  alcohol  may  be  produced  by  butyric  acid  bacteria. 

If  gas  formation  becomes  noticeable  in  milk,  it  is  usually  an 
indication  of  fecal  contamination  ( B . coli,  aerogenes,  more  rarely  B. 
amylobacter) , but  during  hot  summer  weather  the  milk  containers  are 
sometimes  heavily  infested  by  yeasts,  w'hich  may  cause  considerable 
losses  by  the  foaming  of  milk  and  cream  in  transport,  or  by  fermenta- 
tions in  condensed  milk.1  Gas  forming  varieties  of  micrococci,  strepto- 
cocci, and  lactobac-illi  may  also  become  active,  although  they  are  not 
very  numerous.  It  is  worth  knowing  that  part  of  the  mastitis  strepto- 
cocci are  strong  gas  producers ; therefore,  mastitis  milk  does  not  infre- 
quently cause  gas  formation  in  the  fermentation  test,  as  well  as  in  cheese. 
Intestinal  bacteria  (B.  coli  and  aerogenes)  give  a mixture  of  carbon 
dioxide  and  hydrogen,  while  streptococci,  lactobacilli,  and  yeasts  pro- 
duce carbon  dioxide  exclusively. 

Curdling-  of  Milk.  — The  coagulation  of  the  casein  in  milk  is  not 
always  due  solely  to  the  acids  produced  by  bacteria;  rennet  of  bacterial 
origin  may  participate  in  this  process,  sometimes  it  may  even  act  alone. 
If  the  milk  is  rich  in  true  lactic  acid  bacteria,  the  curd  is  solid,  smooth,  of 
porcelain-like  appearance,  and  no  or  little  whey  of  pale  color  is  pressed 
out  (see  Fig.  1,  b,  Plate  X).  The  presence  of  weak  strains  is  usually 
characterized  hy  a larger  amount  of  whey.  Rennet  producing  organ- 
isms make  a soft,  loose  coagulum,  that  shrinks  gradually;  the  whey  is 
of  yellowish,  greenish,  or  brownish  color  (see  Fig.  1,  c,  Plate  X).  Such 
“cheesy”  coagulation  of  milk  is  caused  by  micrococci,  different  non- 
sporulating  bacteria,  such  as  B.  fluorescens,  and  especially  by  sporulat- 
ing bacilli  (B.  subtilis  and  mesentericus) , whose  action  is  sometimes 
very  troublesome  in  pasteurized  and  incompletely  sterilized  milks.  Most 
frequently  acid  and  rennet  combine  their  effects;  many  species  are 
known  to  act  simultaneously  in  both  ways.  The  majority  of  micro- 
cocci are  acid  and  rennet  producers;  streptococci,  non-sporulating  and 
sporulating  bacteria  may  display  the  same  abilities.  Because  such 
micrococci  are  usually  present  in  the  udder,  acid  and  rennet  coagulation 
of  the  milk  is  so  frequent, 

>0.  Hunter,  Jour.  Bad.,  vol.  3,  1918,  p.  293;  B.  W.  Hammer,  Iowa  Agr.  Exp. 
Stat,  Research  Bull.  54,  1919. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


181 


Sometimes  an  abnormally  early  curdling  of  the  milk  is  observed, 
which  is  the  result  of  an  excessive  development  of  acid  and  rennet  pro- 
ducing cocci  within  the  udder.  This  may  be  restricted  to  individual 
cases,  or  all  the  milk  produced  may  undergo  this  abnormal  change,  es- 
pecially on  very  hot  days  with  thunderstorms.  In  the  latter  ease,  the 
effect  caused  by  the  contaminations  within  the  udder  is  usually 
aggravated  by  additional  contaminations  due  to  less  careful  milking 
and  handling  of  the  milk,  as  well  as  to  increased  growth  of  the  microflora 
in  washed  but  not  sterilized  utensils.  However,  even  if  great  care  is 
taken,  and  the  milk  is  thoroughly  cooled  in  order  to  offset  the  influence 
of  the  hot  weather,  the  increased  initial  contamination  may  still  cause 
spoilage.  In  the  production  of  certified  or  Grade  A milk  close  observa- 
tions of  these  fluctuations  of  the  udder  flora  are  very  important.1 

Decomposition  of  Casein. — The  acid  formed  in  normal  milk  prevents, 
as  a rule,  further  decomposition  of  the  casein,  but  whenever  incom- 
pletely sterilized  milk,  or  milk  with  a very  low  initial  germ  content,  is 
kept  for  several  days  or  weeks,  a partial  dissolution  and  disintegration 
of  the  casein  will  occur,  accompanied  by  abnormal  alterations  of  taste 
and  flavor;  occasionally,  even  distinctly  poisonous  substances  are 
produced.  The  last-named  possibility  was  the  reason  why  the  manu- 
facture of  so-called  sterilized  milk  could  not  succeed,  although  it  was 
considered  to  be  the  safest  food  for  children.  Spores  survive  in  such 
milk,  and  if  it  is  kept  for  some  time,  the  newly  grown  bacilli,  working 
under  anaerobic  conditions,  act  very  unfavorably  upon  the  casein. 
The  partial  digestion  which  becomes  visible  under  such  circumstances 
is  not  always  true  peptonization,  since  peptic  enzymes  work  only  if 
the  reaction  is  acid.  A distinctly  alkaline  reaction  is  rather  frequent  in 
these  cases,  and  tryptic  enzymes  are  produced  by  the  bacteria  active  in 
heated  milk. 

A small  amount  of  ammonia  is  always  produced  in  the  course  of 
casein  decomposition.  Several  authors  have  tried  to  develop  a special 
test  upon  this  basis,  and  it  is  indeed  quite  probable  that  low-grade  milk 
will  contain  more  ammonia  than  can  be  found  in  good  milk.  But  no 
satisfactory  method  is  known  at  present  which  would  permit  rapid  and 
accurate  determinations  of  this  kind. 

Alterations  of  the  Milk  Fat. — Sometimes  a partial  decomposition 
of  milk  fat  takes  place  before  the  milk  leaves  the  udder  or  very  soon 
afterwards;  such  milk  is  characterized  by  a more  or  less  rancid  taste 
and  flavor.  The  fat  splitting  variety  of  B.  abortus,  which  is  not  in- 
frequent in  healthy  udders,  may  be  responsible;  several  other  species, 

1 F.  Lohnis,  Molkerei-Zeitg.  Hildesheim,  vol.  28,  1914,  p.  785. 


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such  as  B.  fluorescens  and  related  non-sporulating  rods,  as  well  as  cer- 
tain micrococci,  are  capable  of  acting  in  a similar  manner.  Usually, 
however,  no  pronounced  change  of  the  fat  will  occur  during  the  com- 
paratively short  time  the  milk  is  kept,  perhaps  with  the  only  exception 
that  the  cream  will  be  partially  oxidized,  if  bottled  milk  is  exposed  to 
direct  sun-light.  A “tallowy”  taste  and  flavor  is  produced  in  the  layer 
of  cream. 

Abnormal  Taste  and  Flavor  of  Milk. — Different  kinds  of  abnormal 
tastes  and  flavors  occur  in  freshly  drawn  milk,  mostly  at  the  end  of 
lactation  and  under  the  influence  of  unsuitable  food,  that  are  not  due 
to  bacterial  action;  but  if  the  abnormalities  develop  gradually  after- 
wards, microorganisms  are  always  responsible  for  such  alterations. 
Sometimes  the  changes  can  be  correlated  with  distinct  chemical  trans- 
formations, as  in  the  decomposition  of  casein  and  fat,  but  in  other  cases 
it  is  rather  difficult  to  give  an  exact  definition  of  the  abnormal  flavors. 
Many  species  of  bacteria  have  been  described  which  were  found  active 
in  such  milk,  but  practically  all  of  them  lost  their  specific  characters 
when  they  were  grown  for  a while  on  artificial  substrates.  Most  of 
them  are  to  be  classed  as  varieties  of  B.  coli,  fluorescens,  and  proteus, 
usually  originating  in  the  intestines  and  carried  into  the  milk 
as  fecal  contaminations.  The  special  environmental  conditions  that  pre- 
vail in  the  digestive  tract  under  the  influence  of  different  feeding,  stimu- 
late the  appearance  of  such  modifications  in  the  fecal  flora.  The  more 
fecal  contaminations  are  allowed  to  get  into  the  milk,  the  more  will  the 
effect  of  good  and  bad  feed  become  noticeable  in  the  milk  obtained.  Ac- 
cordingly, abnormal  taste  and  flavor  of  clean  milk  is  due  more  gen- 
erally to  initial  chemical  alterations  than  to  secondary  bacterial  actions. 

Pigmentation  of  Milk. — Pigment  producing  bacteria  have  been 
found  occasionally  within  the  udder,  but  under  these  circumstances  they 
are  unable  to  produce  any  color  in  the  milk.  A reddish  hue,  sometimes 
visible  in  freshly  drawn  milk,  indicates  invariably  the  presence  of  blood 
and  a diseased  condition  of  the  udder.  In  all  other  cases  several  days 
will  elapse  before  such  pigments  will  be  produced  by  bacteria,  as  are 
shown  in  Plate  VI.  It  was  pointed  out  on  p.  89  that  at  the  present 
time  these  organisms  and  their  activities  have  lost  much  of  their  for- 
mer importance,  but  a yellow  color  on  cream  and  a greenish  discolora- 
tion of  milk  are  not  very  rare,  if  milk  is  kept  for  some  time  in  cold 
storage;  micrococci  or  short  rods  are  active  in  the  first  case,  B.  fluor- 
escens in  the  latter. 

If  milk  is  kept  in  rusty  containers,  the  acids  produced  dissolve  part 
of  the  rust.  A grayish-blue  color  and  a disagreeable  taste  are  the  results 
of  this  improper  handling. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


183 


Slimy  and  Ropy  Milk. — Increased  viscosity  that  makes  milk  slimy, 
or  in  extreme  cases  distinctly  ropy,  may  be  due  to  the  growth  of  varie- 
ties of  lactic  acid  bacteria  which  have  lost  part  of  their  ability  to 
produce  acid  and  have  increased  their  inclination  to  construct  slime 
capsules  around  their  cells.  Because  slime  producing  varieties  are 
known  to  occur  in  all  four  groups  of  lactic  acid  bacteria,  they 
are  nearly  as  omnipresent  as  the  typical  lactic  acid  bacteria,  but 
since  their  resistance  against  unfavorable  influences  is  higher  on  ac- 
count of  their  thick  capsules,  they  may  often  survive  in  utensils, 
wherein  the  acid  producers  have  been  suppressed  by  cleaning  that  is 
not  followed  by  sterilization.  The  latter  procedure  kills  all  slime  pro- 
ducers, and  to  heat  all  utensils  thoroughly  by  steam,  or  better  in  a hot 
air  oven,  is  the  only  means  by  which  the  trouble  can  be  promptly 
eliminated.  In  addition,  the  source  of  infection  should  be  investigated; 
in  most  cases  it  is  the  water  or  the  litter.  Adding  small  amounts  of 
the  materials  to  the  milk  kept  at  a suitable  temperature,  leaves,  as  a rule, 
no  doubt  about  the  source  of  infection.  As  long  as  the  ropiness  is  not 
too  pronounced,  the  milk  can  be  used  in  the  household ; it  is  disagree- 
able, but  not  unhealthful.  Sporulating  slime  producers  and  slimy  va- 
rieties of  Oidium  lactis  may  also  occur  in  milk,  but  they  are  by  no 
means  as  common  as  those  varieties  of  lactic  acid  bacteria. 

Elimination  of  Abnormal  Alterations  of  Milk. — Whenever  abnor- 
mal alterations  of  milk  are  to  be  investigated,  it  must  always  be  kept 
in  mind  that  the  chemical  composition  of  that  particular  milk  may  be 
the  main  cause  why  certain  bacteria  grow  so  well  in  it.  It  is  no  rare 
occurrence  that  laboratory  experiments  remain  inconclusive,  because  the 
isolated  strains,  when  tested  in  pure  culture  in  other  milk,  which  was 
perhaps  sterilized  before,  fail  to  display  the  characters  which  they  have 
shown  before.  Local  inspections  and  simple  fermentation  tests  give 
usually  much  more  satisfactory  and  more  useful  results,  than  are  fur- 
nished by  the  isolation  of  “new  species”  in  the  laboratory.  These  so- 
called  species  are  in  most  cases  nothing  but  local  varieties  of  well  known 
bacteria,  which  have  modified  their  character  temporarily,  in  accordance 
with  environmental  conditions.  Spontaneous  appearance  and  disap- 
pearance of  such  abnormalities  find  their  explanation  in  these  facts. 
Utmost  cleanliness,  thorough  sterilization  of  all  utensils,  chlorination  of 
the  water,  and  if  necessary  pasteurization  of  the  milk  are  to  be  recom- 
mended as  practical  remedies. 


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3.  MILK  PASTEURIZATION— FERMENTED  MILKS 

Certain  effects  of  bacterial  activities  in  milk  could  be  prevented  and 
remedied  by  chemical  means.  For  example,  increased  acidity  could  be 
neutralized  by  the  addition  of  sodium  bicarbonate;  or  peroxide  of 
hydrogen  might  be  added  to  the  milk  in  order  to  postpone  its  acidifi- 
cation. In  exceptional  cases  the  use  of  these  two  relatively  harmless 
chemicals  may  be  permissible,  but  as  a rule  it  is  far  preferable  to  rely 
upon  careful  pasteurization  and  refrigeration.  Pure  food  laws  properly 
forbid  the  addition  of  chemicals  to  milk. 

Milk  Pasteurization. — It  was  pointed  out  before  that  the  pasteuri- 
zation of  milk  by  the  application  of  heat  reduces  the  germ  content  and 
thus  increases  the  keeping  quality  of  milk.  The  relatively  best  method 
consists  in  exposing  the  bottled  milk  to  63°  C.  for  20  to  30  minutes, 
then  reducing  its  temperature  by  cold  water  or  cold  air  to  about  5°  C., 
and  afterwards  keeping  it  below  10°  C.  Increasing  the  temperature  to 
75°  or  80°  C.,  as  in  the  so-called  flash  process,  biorization,  etc.,  has  no 
advantage  over  the  pasteurization  at  63°  C.  The  chemical  qualities  of 
the  milk  are  much  less  affected  by  the  latter  method,  but  the  killing  of 
pathogenic  and  of  other  harmful  bacteria  is  just  as  well  realized,  pro- 
vided that  the  temperature  of  63°  C.  is  carefully  maintained  all  the  time. 

Various  tests  are  available  which  permit  to  determine  whether  the 
heating  was  sufficient  for  thorough  pasteurization.  Because  formerly 
the  application  of  75°  to  80°  C.  was  generally  considered  necessary  for 
this  purpose,  most  methods  that  have  been  worked  out  thus  far,  can  be 
used  only  for  the  examination  of  milk  pasteurized  at  such  high  tempera- 
tures. Raw  milk  contains  enzymatic  substances  whose  presence  can  be 
proved  by  certain  color  reactions,  but  after  the  milk  has  been  exposed 
to  75°  to  80°  C.,  these  enzymes  are  more  or  less  inactivated  and  the 
color  tests  give  negative  results.  Para-pRenylen-diamin  and  guaiacol 
are  the  substances  most  frequently  used  for  such  examinations.  How- 
ever, no  biochemical  test  is  known  that  would  be  applicable  to  milk 
pasteurized  at  63°  C.  Only  by  making  microscopical  examinations  is  it 
possible  to  ascertain  promptly  whether  the  heating  was  sufficient  or  not. 
In  raw  milk  the  bacteria  are  easily  stained,  while  the  leucocytes,  and  es- 
pecially their  nuclei,  mostly  refuse  to  take  the  stain,  and  appear  there- 
fore under  the  microscope  white  against  a darker  background.  In  pas- 
teurized milk  the  situation  is  reversed;  many  of  the  bacteria  are  pale, 
while  the  leucocytes  and  their  nuclei  are  darkly  stained,  their  size  is 
generally  reduced,  and  many  of  the  nuclei  have  been  broken  up  in 
several  parts.1 

1 W.  D.  Frost  and  G.  D.  Moore,  Jour.  Dairy  Science,  vol.  2,  1919,  p.  1S9;  W.  D. 
Frost,  Univ.  of  Wis.  Studies  No.  2,  1921,  pp.  151-163  with  plates. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


185 


Proper  pasteurization  kills  approximately  99  per  cent  of  all  bac- 
teria growing  in  milk,  so  that  only  a few  hundreds  or  a few  thousands 
will  survive.  If  such  milk  is  promptly  cooled  and  kept  below  10°  C., 
its  germ  content  should  still  be  low  at  the  time  of  delivery.  In  fact, 
however,  much  of  the  pasteurized  milk  is  very  rich  in  bacteria  when 
it  gets  into  the  hands  of  the  consumer,  and,  as  was  pointed  out  before, 
this  secondary  growth  of  organisms  is  quite  different  from  and  much 
more  objectionable  than  the  original  microflora.  The  application  of  the 
methylene  blue  reduction  test  makes  the  bacteriological  examination  of 
pasteurized  milk  a relatively  easy  matter,  and  it  seems  very  desirable 


fllayofirm-Yoghurt 

Dr.  Lttloff  6c  Dr.  (Tlaver,  Breslau  13 


LABORATORIUM  MOSER 


(Tlavofirm-Voghutfl- milch 

Die  Milch  alsjtngonjiinci)  jfcr  ■■ 


,Vihdobono"-Yoghurt-  end  Kefir-Reinkulturcn 


Fig.  45.— Yaourt  cultures  in  powder  and  liquid  form  Q nat.  size). 


that  extensive  use  be  made  of  this  simple  method,  in  order  to  eliminate 
all  so-called  pasteurized  milk  whose  short  reduction  time  would  indi- 
cate that  it  has  been  improperly  handled. 

Fermented  Milks. — Only  a small  number  of  lactic  acid  bacteria 
11  survive  pasteurization ; therefore  it  has  repeatedly  been  recommended 
to  add  pure  cultures  of  lactic  acid  streptococci  to  such  milk.  A fairly 

I normal  microflora  would  thus  be  restored  and  good  sour  milk  could  be 
prepared,  if  desired.  In  Eastern  European  and  in  Asiatic  countries, 
where  lack  of  cleanliness  and  high  summer  temperatures  invite  early 
; spoilage  of  all  milk,  similar  procedures  have  long  been  in  use.  The  milk 


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is  boiled,  sometimes  for  several  hours,  and  then  inoculated  with  a smali 
amount  of  fermented  milk,  or  with  special  preparations  which  have  been 
found  useful  for  this  purpose.  Because  the  milk  has  to  stand  rather 
high  air  temperatures,  the  original  material  used  for  inoculation  is 
taken  quite  properly  from  the  stomachs  of  milk-fed  lambs  and  calves, 
where  lactobaeilli  adapted  to  relatively  high  temperatures  grow  almost 
in  pure  culture.  As  usual,  some  symbiotic  yeasts  accompany  the  laeto- 
bacilli,  and  they  contribute  by  their  alcohol  production  to  the  particu- 
lar flavor  of  these  types  of  fermented  milks.  One  of  them,  the  so-called 
yaourt  or  yoghurt  of  Bulgaria,  Roumania,  Greece,  and  Turkey  is  now 
widely  used  in  Europe  and  in  America.  Some  of  the  cultures  offered 
by  the  trade  are  shown  in  Fig.  45;  frequently  they  are  of  in- 
ferior quality.  Sour  milk  prepared  along  the  same  lines  is  called 
dadhi  in  British  India,  matzoon  in  Armenia,  lebben  in  Syria,  Egypt,  and 


Fig.  46. — Kefir  granules  (I  nat.  size).  Two  1-g.  samples  dry  (a  and  c) 
and  fresh  (6  and  d). 


Algiers,  gioddu  or  mezzoradu  in  Sicily,  cieddu  in  Sardinia,  grusavina 
in  Montenegro,  huslanka  in  the  Carpaths.  Lactobacilli,  lactic  acid 
streptococci,  and  yeasts  are  always  present  and  active;  local  varieties 
are  responsible  for  the  slight,  but  characteristic  differences  in  taste  and 
flavor. 

If  large  quantities  of  fermented  milk  are  consumed  regularly,  as  is 
done  by  those  primitive  people,  the  lactic  acid  bacilli  will  suppress  most 
other  bacteria  in  the  intestines;  putrefaction  and  toxin  production  will 
cease  almost  completely,  and  frequently  an  improvement  in  general 
health  will  result.  But  it  is  not  so  much  the  bacteria  introduced  with 
the  milk,  as  the  milk  itself  that  acts  favorably.  Babies,  calves,  and 
lambs  are  not  fed  fermented  milks,  and  yet  their  digestive  tracts  con- 
tain hardly  anything  else  than  lactobaeilli.  Milk  inoculated  with  calves’ 
feces  and  kept  at  45°  C.,  gives  after  a few  transfers  a “yaourt,”  as 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  MILK 


187 


good  as,  or  better  than  any  one  imported  from  the  East.  Accordingly, 
it  has  also  been  tried  to  improve  the  intestinal  flora  in  man  by  feeding 
pure  cultures  of  B.  acidophilus,  that  is,  a lactobacillus  variety  most 
common  in  the  digestive  tract  of  milk-fed  babies.  But  again  it  was  ob- 
served that  the  desired  change  was  secured  only  if  regularly  large  quan- 
tities of  milk  or  of  milk  sugar  were  consumed,  and  in  this  case  B.  acido- 
philus establishes  itself  spontaneously,  as  it  does  in  every  child. 

There  are  a few  other  fermented  milks  which  have  also  been  recom- 
mended for  therapeutic  purposes.  They  are  the  koumiss  and  the  kefir,, 
both  of  Russian  origin  and  both  characterized  by  their  relatively  high 
content  of  alcohol  (1  to  2 per  cent)  and  of  carbon  dioxide.  Again 
streptococci,  lactobacilli,  and  yeasts  are  working  together,  but  the  latter 
are  much  more  active  in  these  cases.  Genuine  koumiss  is  always  made 
of  mares  ’ milk  which  is  relatively  rich  in  sugar,  and  therefore  especially 
liable  to  undergo  an  alcoholic  fermentation.  By  distilling  koumiss 
highly  intoxicating  liquors  are  manufactured  by  Siberian  tribes.  The 
microorganisms  of  kefir  form  quaint  berry-like  agglomerations,  which 
have  been  widely  sold  in  Europe  and  in  America  when  the  use  of  kefir 
was  in  vogue.  In  Fig.  46  they  are  shown  in  the  dry,  shrunken  condition 
as  they  come  in  the  trade,  and  in  the  swollen,  soaked  state  which  they 
assume  in  milk. 

A third  type  of  fermented  milk  is  prepared  in  the  Scandinavian 
countries,  in  Holland,  and  in  Northern  France,  where  the  air  is  gen- 
erally cool,  and  the  streptococci  are  therefore  more  inclined  to  overgrow 
the  lactobacilli  and  yeasts,  although  these  too  are  present.  The  variety 
of  streptococci  predominating  in  these  milks  produce  lactic  acid,  as  well 
as  fairly  large  quantities  of  slime,  and  these  milks  show  therefore,  after 
they  have  curdled,  a peculiar  “tight,”  gelatinous  consistency,  which  is 
the  reason  why  they  are  called  in  Norway  and  Sweden  taette  and  taet- 
temjolk,  that  is,  tight  milk.  The  fiili  or  puma  prepared  by  Finnish 
settlers  in  Minnesota  is  another  example  of  this  type  of  sour  milk. 


CHAPTER  X 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  BUTTER 

Butter  contains  on  the  average  80  or  more  per  cent  of  milk  fat,  about 
16  per  cent  of  water,  and  small  quantities  of  casein  and  of  salt,  if  this 
is  added.  The  salt  used  is  equivalent  to  2y2  to  3 per  cent  of  the  total 
weight;  it  dissolves  in  the  water  and  changes  this  to  a rather  concen- 
trated brine.  Pure  fat,  such  as  lard  or  other  commercial  products,  is 
almost  completely  resistant  against  attacks  of  microorganisms,  because 
they  can  not  live  on  fat  alone.  If  butter  is  melted  and  carefully  freed 
from  all  non-fat  by  skimming  and  decanting,  the  purified  butter  fat  may 
be  kept  practically  unchanged  for  many  months.  In  normal  but- 
ter, however,  the  casein  supplies  the  necessary  nitrogenous  food  for 
numerous  microorganisms,  and  if  they  are  checked  by  the  added  salt  or 
by  low  temperatures,  their  enzymes  will  continue  to  act  favorably  or 
unfavorably  upon  taste  and  flavor  of  the  butter.  Extensive  investiga- 
tions of  the  last  twenty  or  thirty  years  have  made  it  clear  that  the  care- 
ful control  and  regulation  of  the  microflora  of  the  butter  is  of  great 
practical  importance.1 * Ill, 


1.  GERM  CONTENT  OF  BUTTER 

The  germ  content  of  butter  may  be  low  or  high,  because  it  is  influ- 
enced by  several  circumstances  that  are  highly  variable.  The  micro- 
flora  of  the  cream  is  of  prominent  importance,  which  in  its  turn  is  de- 
pendent on  the  milk,  and  on  the  changes  occurring  during  separation, 
transportation,  pasteurization,  and  ripening  of  the  cream.  The  methods, 
materials,  and  utensils  used  for  butter-making  add  their  specific  influ- 
ences. Careless  treatment,  impure  water,  low-grade  salt,  and  unclean 
utensils  will  necessarily  injure  the  quality  of  the  product.  Further 
changes  in  the  microflora  of  the  butter  take  place  while  it  is  stored  or 
shipped,  and  when  the  butter  reaches  the  consumer  many  alterations 
may  be  noticed  which  could  have  been  prevented,  if  proper  attention 
had  been  paid  to  the  possible  effects  of  bacterial  action. 

1 For  references  see  F.  Lohnis,  “Handbuch  der  landw.  Bakteriologie,”  Chap. 

Ill,  2. 


1S8 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  BUTTER  189 


Microorganisms  in  Cream,  Water  and  Salt. — Because  of  their  rela- 
tively large  size  and  great  number,  the  fat  globules  in  milk  carry 
comparatively  large  quantities  of  bacteria  into  the  cream ; accord- 
ingly, the  germ  content  of  cream  is  always  higher  than  that  of  the 
milk  used.  Pasteurization  kills  approximately  99  per  cent,  but  rapid 
multiplication  takes  place  in  the  ripening  process.  Hundreds  of  mil- 
lions of  bacteria  are  regularly  present  in  ripened  cream,  and  if  this  is 
allowed  to  become  very  acid,  one  or  several  thousand  millions  will  be 
found,  mostly  lactic  acid  bacteria,  but  always  accompanied  by  a variable 
number  of  other  species. 

The  ivater  used  for  rinsing  the  utensils  and  for  washing  the  butter, 
should  be  boiled  or  otherwise  sterilized,  unless  its  germ  content  is  un- 
usually low.  B.  fluorescens,  slime  producing  bacteria,  such  as  Bact. 
lactis  viscosum,  and  spores  of  B.  amylobacter  are  common  in  water, 
and  if  they  get  into  the  butter  in  considerable  quantities,  a distinctly 
unfavorable  effect  will  result. 

Salt,  too,  is  sometimes  very  rich  in  microorganisms  wdiieh  are  able  to 
withstand  the  antiseptic  effect  of  the  sodium  chloride,  and  may  cause  a 
marked  deterioration  of  the  quality  of  the  butter.  Only  salt  should 
be  used  that  is  absolutely  pure,  baeteriologically  as  well  as  chemically; 
chemical  impurities,  such  as  traces  of  iron  compounds,  may  act  as  un- 
favorably upon  the  taste  of  the  butter,  as  may  be  the  case  with  micro- 
organisms. 

Influence  of  Utensils,  Air,  and  Paper. — Aside  from  the  microor- 
ganisms brought  into  the  butter  with  the  materials  used  in  its  manu- 
facture, other  forms  are  added  by  the  contact  with  baeteriologically  un- 
clean utensils.  Metal  is  not  very  suitable  for  churns,  etc.,  because  the 
delicate  flavor  of  butter  is  easily  impaired  in  such  containers ; wooden 
churns  and  utensils  are  generally  preferred,  despite  the  fact  that  they 
are  more  difficult  to  clean  and  to  sterilize.  For  such  churns  the  appli- 
cation of  dry  heat  is  impossible,  and  steaming  proves  also  detrimental 
to  the  structure  of  the  wood.  Some  kind  of  chemical  treatment  must 
be  relied  upon ; milk  of  lime  has  proved  suitable  for  this  purpose. 

The  air  in  rooms  where  butter  is  made  and  kept,  should  be  free  of 
microorganisms  which  might  grow  on  the  butter  and  injure  its  quality. 
Mold  spores  come  first  in  this  respect ; if  necessary,  they  should  be  killed 
by  fumigation. 

The  paper  used  for  wrapping  the  butter  may  act  favorably  or  un- 
favorably upon  its  quality.  Good  paper  protects  the  butter  against  the 
invasion  of  molds,  but  paper  of  inferior  quality,  that  is,  paper  which  is 
comparatively  rich  in  soluble  organic  substances,  such  as  dextrin  and 
glycerol,  favors  the  growth  of  microorganisms  upon  the  butter,  even  if 


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it  does  not  itself  carry  additional  contaminations.  Previous  boiling  of 
the  paper  in  a 25  per  cent  brine  removes  such  substances,  and  at  the 
same  time  kills  all  microorganisms  that  may  adhere  to  the  paper. 

Changes  in  Germ  Content  During  Storage. — If  butter  is  made  from 
pasteurized  sweet  cream  its  initial  germ  content  is  low,  but  within  the 
first  few  days  a rapid  increase  takes  place,  due  to  the  multiplication  of 
lactic  acid  bacteria,  which  is  followed  by  a gradual  decline.  If  ripened 
cream  is  used,  the  lactic  acid  bacteria  have  already  grown  therein,  and 
the  maximal  germ  content  is  reached  at  the  beginning;  only  in  cases 
where  the  microflora  of  the  sour  cream  was  very  mixed,  a slight  in- 
crease of  the  total  number  may  occur  during  the  first  few  days.  The 
gradual  dying  of  the  microorganisms  in  old  butter  is  always  noticeable. 
The  following  figures  represent  typical  counts: 


Millions  per  g.  Butter 

Freshly  Made 

After  1 Week 

After  2 Months 

Butter  r pasteurized  sweet  cream 

1 

10-30 

0.6-2 

made  1 ripened  cream 

10 

3-  8 

0.1-2 

from  l impure  sour  cream 

20 

5-40 

0.4-2 

The  lower  figures  recorded  after  1 week  and  2 months  were  obtained 
with  material  taken  from  inner  parts,  whereas  the  higher  figures  always 
refer  to  the  bacterial  growth  on  the  surface  of  the  butter.  The  micro- 
organisms active  in  the  decomposition  of  the  fat  are  all  aerobic  ; the  casein 
which  they  also  attack  furnishes  ammonia  and  other  products  of  alkaline 
reaction,  and  the  resulting  partial  neutralization  of  the  acidity  favors  the 
continued  growth  of  the  lactic  acid  bacteria,  which  are  rapidly  killed  by 
the  acid  in  the  inner  part  of  the  butter. 

If  the  butter  has  not  been  thoroughly  freed  from  casein,  its  germ 
content  may  reach  100  millions  per  gram  and  more,  while  the  best 
grade  of  butter  made  for  the  export  trade  is  characterized  by  excep- 
tionally low  bacterial  counts.  The  absence  of  air  in  the  containers  pre- 
vents any  rise  in  numbers.  For  example,  the  following  counts  were 
obtained  from  such  material : 

Microorganisms  After  1 2 3 18  weeks 

in  1 g.  butter  362,000  125,000  23,600  200 

Types  of  Butter  Organisms. — Almost  without  exception  lactic 
acid  bacteria  make  up  the  vast  majority  of  butter  organisms.  Among 
them  nearly  always  the  streptococci  predominate,  especially  if  the 
cream  was  ripened  at  a temperature  in  the  neighborhood  of  20°  C.  If 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  BUTTER  191 


it  was  kept  at  lower  degrees,  the  lactic  acid  micrococci  become  more 
conspicuous ; the  same  holds  true  in  regard  to  the  influence  of  cold 
storage.  Lactobacilli  are  also  present ; they  grow  slowly  in  the  butter, 
but  after  a few  weeks  they  may  survive  the  short-lived  streptococci. 
Low-grade  butter  is  comparatively  rich  in  B.  coli  and  aerogenes. 

Other  non-sporulating  and  sporulating  bacteria,  such  as  B.  fluor- 
escer.s,  B.  amylobacter,  subtilis,  and  mesentericus,  are  found  quite  regu- 
larly, though  not  in  great  numbers.  Their  influence  upon  the  quality 
of  the  butter  is  sometimes  very  marked. 

Yeasts,  molds,  and  actinomycetes  are  rather  common  in  butter  of 
inferior  quality.  A marked  increase  in  yeasts  is  frequently  noticeable 
in  old  butter.  Molds  were  found  to  be  more  numerous  in  margarine 
than  in  butter. 

Pathogenic  microorganisms  may  be  carried  by  butter,  as  well  as 
by  milk,  but  thorough  pasteurization  of  the  cream  and  strict  ob- 
servance of  all  rules  concerning  the  prevention  of  the  spreading  of 
pathogenic  organisms,  especially  with  regard  to  healthy  disease  car- 
riers, will  eliminate  this  possibility.  Grass  bacilli  are  not  infrequent 
in  butter,  where  they  are  sometimes  mistaken  for  tubercle  bacilli. 

2.  BACTERIAL  ACTION  AND  QUALITY  OF  BUTTER 

Taste  and  flavor  of  butter  is  dependent,  in  the  first  place,  upon  the 
quality  of  milk  and  cream.  It  should  be  remembered  that  the  specific 
influence  exerted  by  different  foodstuffs  is  frequently  more  pronounced 
in  butter  than  in  milk,  because  the  milk  fat  is  much  more  easily  affected 
than  are  the  other  milk  constituents.  “Grass  butter”  made  in  sum- 
mer is  rather  different  from  the  “straw  butter”  produced  in  winter; 
the  effects  of  good  and  bad  silage,  of  beet  tops,  and  of  some  of  the 
concentrated  foodstuffs  are  very  marked.  Nevertheless,  good  butter  can 
be  made  from  all  such  milk,  provided  that  the  cream  is  thoroughly 
pasteurized,  well  ripened,  and  the  butter  is  carefully  made.  Slight 
changes  in  the  treatment  of  cream  and  butter  may  help  to  reduce  or  to 
eliminate  certain  unfavorable  influences  of  the  milk ; the  microorganisms 
growing  in  cream  and  butter  are  used  in  this  case  for  improving  the 
quality  of  the  butter.  However,  there  are  other  possibilities  where  bac- 
teria or  fungi  may  act  unfavorably  upon  the  butter,  and  their  enzymes 
may  continue  these  harmful  activities  after  the  organisms  themselves 
have  died. 

Influence  of  Microorganisms  upon  Taste  and  Flavor. — When  pure 
cultures  of  lactic  acid  bacteria  were  first  used  for  ripening  pasteurized 
cream,  the  objection  was  frequently  raised  that  such  butter  was  lacking 


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in  flavor,  although  its  better  keeping  quality  was  generally  acknowl- 
edged. Naturally,  the  mixed  microflora  growing  in  home-made  starters, 
caused  a much  more  varied  activity  in  the  butter,  resulting  in  a stronger 
flavor,  but  at  the  same  time  in  a quicker  deterioration  of  the  product. 
Gradually  the  cleaner  though  milder  taste  and  flavor  of  butter  made 
with  pure  cultures  was  more  and  more  appreciated.  Attempts  to  add 
special  types  of  aroma  producing  microorganisms  to  the  starters  have 
been  made  repeatedly,  but  most  of  these  experiments  were  unsuccessful; 
for  a while  the  selected  strains  continued  to  act  favorably,  sooner  or 
later,  however,  they  changed  and  began  to  show  distinctly  unfavorable 
features.  Permanently  satisfactory  results  have  been  secured  only  with 
certain  strains  of  lactic  acid  streptococci,  of  which  it  was  first  shown  by 
the  Danish  dairy  expert  Storeh  that  they  may  exert  either  none,  or  a 
favorable,  or  an  unfavorable  effect  upon  taste  and  flavor  of  the  butter. 
Volatile  acids  produced  from  the  lactates  first  formed,  or  from  the  cit- 
rates originally  present  in  milk  and  cream,  are  largely  responsible  for 
the  desired  flavor  of  butter.1  Skillful  selection  and  propagation  of 
good  strains  of  such  organisms  have  become  important  tasks  of  dairy 
bacteriologists ; the  great  and  often  unexpected  influence  of  symbiotic 
action  must  also  be  carefully  considered  in  such  cases. 

Abnormal  Flavors. — Some  strains  of  lactic  acid  streptococci  pro- 
duce peculiar  oily  tastes  in  butter,  as  was  pointed  out  by  Storeh  in 
Denmark  about  thirty  years  ago.  More  frequently,  however,  such  ab- 
normalities as  oily,  fishy,  and  rancid  taste  and  flavor  are  due  to  the 
decomposition  of  the  butter  fat.  Liberation  of  trimethyl-amine  from 
lecithin  may  act  as  another  cause  of  fishy  flavor.2  A peculiar  “cheesy” 
or  “sour”  taste  is  sometimes  created  by  a heavy  growth  of  laetobaeilli, 
which  is  frequent  in  cream  that  was  not  promptly  cooled  after  pas- 
teurization. As  usual,  yeasts  are  simultaneously  present  in  such  but- 
ter; if  their  number  is  large,  a “yeasty”  flavor  will  appear.  If  the 
casein  is  strongly  attacked  by  proteolytic  bacteria  a distinctly  bitter 
taste  may  become  noticeable,  or  it  may  be  merely  more  or  less  “impure,” 
when  the  decomposition  has  made  less  progress.  The  longer  the  butter  is 
kept,  the  more  will  its  flavor  deteriorate,  due  to  the  combined  action  of 
microorganisms  and  of  their  enzymes  upon  all  the  constituents  of  the 
butter. 

Thorough  pasteurization  of  the  cream  and  inoculation  with  pure 
cultures  of  lactic  acid  bacteria  will  prevent  and  retard  many  of  these 
undesirable  changes.  But  since  new  contaminations  with  such  common 

1B.  W.  Hammer,  Iowa  Agr.  Exp.  Stat.  Research  Bull.  63,  1920. 

2 G.  C.  Supplee,  Cornell  Agr.  Exp.  Stat.  Memoir  29,  1919. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  BUTTER  193 

organisms  as  B.  fluoreseens,  Oidium  lactis,  Penicillium  glaucum,  and 
other  molds,  all  of  which  act  unfavorably  upon  the  butter,  can  not  be 
avoided  entirely,  special  methods  must  be  applied  in  order  to  pro- 
tect the  butter  which  is  not  consumed  within  a short  time.  In  addition 
to  the  removal  of  the  milk  proteins  by  washing,  the  addition  of  salt 
plays  an  important  role  in  this  respect.  Butter  containing  2^  to  3 per 
cent  salt  is  almost  completely  fortified  against  the  fat-splitting  B. 
fluoreseens  and  many  other  bacteria;  the  fungi,  however,  will  continue 
to  grow,  if  they  are  not  checked  by  the  absence  of  air  and  by  the  applica- 
tion of  low  temperatures,  as  in  cold  storage. 

Deterioration  of  Butter  in  Cold  Storage. — At  a temperature  close 
to  0°  C.  psychrophilic  bacteria  are  still  active;  they  die  slowly  at  lower 
temperatures,  but  the  enzymes  produced  by  bacteria  and  fungi  may  con- 
tinue to  act.  If  the  butter  to  be  placed  in  cold  storage  is  carefully 
made  from  thoroughly  pasteurized  sweet  cream,  none  or  very  little  de- 
terioration is  to  be  expected.  Ripened  cream  always  carries  enough 
bacterial  enzymes  to  cause  sooner  or  later  marked  changes  in  taste  and 
flavor,  and  if  traces  of  metal  had  been  dissolved  by  the  acids  a disagree- 
able metallic  flavor  will  appear.  Furthermore,  such  traces  of  metal 
may  hasten  the  deterioration  of  the  butter  by  stimulating  the  enzymatic 
action;  in  this  respect  copper  salts  have  proved  most  effective.1  Spon- 
taneous oxidation  of  fat  and  milk  sugar  contributes  to  the  inferior  flavor 
of  cold  storage  butter,  if  it  is  kept  at  a temperature  close  to  0°  C. ; but 
at  very  low  degrees  ( — 18°  C.)  it  is  practically  of  no  influence.2 

Rancidity  of  Butter. — If  butter  is  made  without  pasteurization  of 
the  cream  and  without  pure  culture  starters,  its  very  mixed  microflora 
always  contains  many  fat-splitting  organisms  that  will  quickly  cause 
rancidity,  especially  if  the  temperature  is  high.  Micrococci,  B.  fluor- 
escens,  prodigiosus,  and  many  molds  are  active  in  such  butter;  among 
the  latter  a species  called  Cladosporium  butyri  is  most  characteristic  of 
rancid  butter.  All  these  organisms  are  aerobic,  and  they  attack  fat 
as  well  as  casein;  accordingly,  they  are  most  active  at  the  surface  of 
the  butter.  The  zone  occupied  by  them  is  marked  by  its  darker,  more 
transparent  appearance,  due  to  the  disintegration  of  the  casein. 

Because  of  the  complex  nature  of  butter  fat  different  volatile  and 
non-volatile  fatty  acids,  as  well  as  glycerol,  are  liberated  by  the  fat-split- 
ting organisms.  The  glycerol  is  quickly  transformed  by  the  same  or 
by  other  bacteria  and  fungi,  and  various  intermediate  products  appear. 
Of  these,  butyric  acid  esters  are  typical  constituents  of  rancid  butter. 

1 O.  F.  Htjnziker  and  D.  F.  Hosman,  Jour.  Dairy  Science,  vol.  1.  1917,  p.  320. 

2 D.  C.  Dyer.  Jour.  Agr.  Research,  vol.  6,  191fi.  p.  927. 


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The  free  acids  present  in  such  butter  can  not  be  accepted  as 
an  accurate  measure  of  its  rancidity,  although  frequently  investigations 
upon  this  subject  have  been  restricted  to  such  determinations. 

Decomposition  and  Oxidation  of  Fat. — When  butter  becomes  ran- 
cid the  bacterial  decomposition  of  the  fat  is  frequently  accompanied  by 
a spontaneous  oxidation  of  the  fat.  For  a long  time  both  processes 
have  not  been  properly  separated,  and  many  incorrect  ideas  have  been 
promulgated.  Oxidation  takes  place  even  with  the  purest  sample  of 
fat  if  this  is  exposed  to  higher  temperature  or  to  direct  sunlight  in  the 
presence  of  air.  If  a piece  of  fresh  butter  is  exposed  to  sunlight,  it  can 
be  easily  ascertained  that  this  purely  chemical  process  leads  to  results 
quite  different  from  those  produced  in  rancid  butter  by  the  fat-splitting 
microorganisms.  If  part  of  the  butter  is  protected  by  wrapping  paper, 
the  alterations  of  the  exposed  part  will  be  clearly  noticeable. 

The  color  of  oxidized  butter  is  lighter,  not  darker  as  in  rancid  but- 
ter; the  taste  is  “tallowy,”  not  rancid;  the  splitting  of  fat  and  the 
transformation  of  glycerol  are  strong  in  rancid,  but  very  weak  in  oxi- 
dized butter ; butyric  acid  esters  are  present  in  rancid  butter,  while 
in  tallowy  butter  aldehydes  are  frequent.  The  white  color  and  the 
abnormal  taste  of  oxidized  butter  are  sometimes  so  conspicuous  that 
occasionally  such  butter  has  been  declared  to  be  adulterated  with  other 
fats.  Low  temperatures  and  the  absence  of  air  prevent  spontaneous 
oxidation,  as  well  as  the  bacterial  decomposition  of  butter  fat. 

Abnormal  Consistency  and  Discoloration  of  Butter. — If  butter  is 
unusually  soft  or  otherwise  of  abnormal  consistency,  the  cause  will  be 
found,  as  a rule,  in  the  application  of  faulty  methods.  Occasionally,  how- 
ever, slime  producing  and  proteolytic  organisms  are  so  numerous  in  the 
cream  that  difficulties  arise  when  the  butter  is  made,  and  the  product 
obtained  is  of  soft  texture  and  of  poor  keeping  quality.  Proper  pasteuri- 
zation will  eliminate  this  trouble.  Sometimes  a rapid  deterioration  of 
butter  has  been  found  to  be  due  to  a heavy  growth  of  anaerobic  butyric 
acid  bacteria;  the  gas  produced  by  these  organisms  makes  numerous  small 
holes  in  the  butter,  looking  like  pin  pricks.  The  spores  of  these  organ- 
isms can  not  be  killed  by  pasteurization,  but  since  they  do  not  germinate 
in  an  acid  substrate,  vigorous  lactic  acid  formation  serves  as  a remedy. 
However,  the  use  of  clean  milk  and  clean  water  will  prevent  contamina- 
tions of  such  kind. 

The  color  of  butter  is  always  dependent  on  the  natural  color  of  the 
milk  fat,  which  varies  according  to  the  food  of  the  cow.  Abnormal  pig- 
mentation, such  as  yellow,  red,  green,  brown,  and  almost  black  discolora- 
tions, may  be  caused  by  various  contaminating  microorganisms,  mostly 
yeasts  and  molds,  which  usually  can  be  excluded  without  great  difficulty. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  BUTTER  195 


Paper  and  containers  are  generally  the  source  of  such  growth;  it  was 
mentioned  before  how  paper  of  unsatisfactory  quality  should  be  treated. 
Irregular  white  spots  and  stripes,  sometimes  visible  in  the  inner  parts 
of  butter,  are  not  caused  by  bacterial  or  fungous  growth,  but  by  an 
irregular  distribution  of  salt  and  water.1 

3.  CREAM  PASTEURIZATION— USE  OF  STARTERS 

It  is  undoubtedly  possible  to  get  satisfactory  results  without  pas- 
teurization of  the  cream  and  without  the  use  of  a high-grade  starter. 
However,  the  results  obtained  in  this  way  may  also  be  very  unsatis- 
factory. The  best  mechanical  treatment  of  the  butter  is  not  sufficient 
to  secure  regularly  a product  of  uniform,  high  quality;  for  this  purpose 
the  biological  requirements  must  be  fully  considered. 

Cream  Pasteurization. — The  application  of  high  temperatures  (85° 
to  90°  C.),  which  can  not  be  recommended  for  the  pasteurization  of 
milk,  is  very  suitable  for  the  pasteurization  of  cream  in  the  making  of 
butter  because  of  the  following  reasons.  In  the  first  place,  the  acci- 
dental microflora  of  the  cream  is  usually  very  mixed,  and  it  is  desirable 
to  eliminate  it  as  completely  as  possible.  In  the  second  place,  the  high 
temperatures  inactivate  the  bacterial  enzymes  which  otherwise  would  con 
tinue  their  harmful  activities  in  the  butter,  if  this  is  placed  in  cold 
storage.  The  relatively  high  percentage  of  fat  and  non-fat  solids  in  the 
cream  reduces  to  some  extent  the  efficiency  of  pasteurization ; it  was  ob- 
served, for  instance,  that  part  of  the  lactobacilli  may  survive  the  tem- 
perature of  90°  C.  in  cream,  while  they  are  promptly  killed  in  milk,  if 
this  is  pasteurized  at  such  high  degrees.  However,  comparatively  few 
lactic  acid  bacteria  will  survive  this  treatment,  and  it  is  absolutely  neces- 
sary,  in  order  to  restore  the  proper  microflora  to  cream  and  butter,  that 
a good  home-made  starter,  or  one  prepared  from  a commercial  culture, 
is  added  to  the  pasteurized  cream.  Without  this  protection  cream  and 
butter  would  be  open  to  the  invasion  of  many  detrimental  organisms. 
It  has  been  tried  repeatedly  to  replace  the  lactic  acid  bacteria  by  adding 
pure  lactic  acid  or  a mixture  of  different  acids  to  the  pasteurized  cream, 
but  the  results  obtained  were  very  unsatisfactory. 

Use  of  Starters. — Sour  milk  or  butter  milk  of  clean  taste  and  flavor 
have  long  served  and  are  still  used  as  home-made  starters,  but  at  present 
it  is  generally  more  satisfactory  to  prepare  pure-culture  starters  by 
inoculating  pasteurized  skim  milk  or  milk-powder  solution  with  a reliable 
commercial  culture.  As  shown  in  Fig.  47,  these  cultures  are  sold  either 

1 0.  F.  Hunziker  and  D.  F.  Hosman,  Jour.  Dairy  Science , vol.  3,  1920,  pp.  77-106 


196 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


in  liquid  form  or  as  a dry  powder.  Liquid  cultures  are  usually  purer 
and  more  active,  but  less  durable  than  those  in  powder  form,  therefore, 
the  former  are  preferable  for  immediate  use,  while  the  latter  may  be  kept 
for  emergencies.  The  labels  of  the  cultures  should  always  show  the  date 
when  the  cultures  have  been  made,  so  that  the  buyer  is  sure  to  receive  a 
fresh,  vigorous  culture;  the  directions  furnished  with  the  cultures  should 
be  followed  carefully.  Most  cultures  contain,  besides  contaminations, 
nothing  but  lactic  acid  streptococci ; by  mixing  strains  that  act  favorably 
upon  taste  and  flavor  of  the  butter  very  satisfactory  results  may  be  se- 


Fig.  47. — Commercial  cultures  for  cream  ripening. 


cured.  In  some  cases  yeasts  are  a regular  constituent  of  the  commercial 
culture;  they,  too,  are  supposed  to  improve  the  flavor  of  cream  and 
butter. 

The  catalase  test,  of  which  it  was  said  (p.  174)  that  it  is  not  of 
much  value  for  the  bacteriological  control  of  milk,  can  be  used  as  a 
simple  and  reliable  means  of  testing  the  purity  of  starters,  because  the 
lactic  acid  streptococci  display  hardly  any  catalytic  abilities.  Strong 
gas  formation  in  the  test  indicates  the  presence  of  contaminations,  which 
should  be  checked  before  they  have  time  to  establish  themselves  in  the 
mother  starter  and  to  act  unfavorably  upon  cream  and  butter. 


CHAPTER  XI 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE 

In  milk  a very  low  germ  content  is  most  desirable;  the  quality  of 
butter  is  best  if  its  microflora  is  composed  almost  entirely  of  lactic  acid 
streptococci ; but  in  cheese  always  very  many  and  very  different  micro- 
organisms are  to  be  found,  because  they  participate  actively  in  the 
various  biochemical  processes  which  in  their  entirety  constitute  the  so- 
called  ripening  of  cheese. 

The  many  kinds  of  cheeses  which  are  on  the  market  owe  their  dif- 
ferences to  the  fact  that  the  special  technique  applied  in  each  case  creates 
environmental  conditions  which  favor  certain  associations  of  microorgan- 
isms whose  activities  produce  the  characteristic  quality,  taste  and  flavor 
of  the  cheese.  It  is  easily  understood  that  curd  taken  from  sour  milk 
presents  conditions  rather  different  from  those  prevailing  in  curd  that 
is  prepared  by  adding  rennet  to  sweet  milk.  The  high  water  content  in 
soft  cheese  and  the  relatively  low  percentage  of  moisture  in  hard  cheese 
influence  the  bacterial  action  very  markedly.  In  small  flat  cheeses,  like 
those  of  Camembert  and  Brie,  the  conditions  are  more  aerobic,  while  they 
are  distinctly  anaerobic  in  the  large  heavy  loaves  of  Swiss  or  Emmental 
and  Cheddar  cheese.  The  special  technique  in  the  preparation  of  milk 
and  rennet,  in  the  treatment  of  the  curd,  in  forming,  pressing,  and  curing 
the  cheese  favors  certain  and  suppresses  other  groups  of  microorganisms. 
Much  valuable  knowledge  upon  all  these  problems  has  been  accumulated 
during  the  last  thirty  years,1  but  many  special  problems  require  con- 
tinued investigations  in  order  to  secure  a completely  satisfactory 
scientific  basis  for  the  much-varied  practice  in  making  and  curing  cheese. 

1.  GERM  CONTENT  OF  CHEESE 

Examination  with  the  naked  eye  shows  that  in  hard  cheeses  of 
Cheddar  and  Emmental  type  no  growth  of  microorganisms  becomes 
visible;  they  contain  nothing  but  bacteria.  In  soft  cheeses,  however, 
various  fungi  may  be  seen  upon  the  surface,  as  on  Camembert  and  Brie, 

'For  detailed  references  see  F.  Lohnis,  “Handbuch  der  landwirtschaftlichen 
Bakteriologie,”  Chap.  Ill,  3. 


197 


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or  inclosed  within  the  curd,  as  in  Roquefort,  Stilton,  and  Gorgonzola. 
Their  number  and  kind  depend  originally  on  the  germ  content  of  the 
materials  and  utensils  used  for  preparing  the  cheese,  but  many  secondary 
alterations  occur  during  the  time  in  which  the  cheese  is  cured ; the  more 
time  is  consumed,  the  greater  is  the  reduction  in  living  cells. 

Origin  of  Cheese  Organisms. — Milk  and  home-made  rennet,  if  this  is 
used,  are  the  main  sources  of  the  microflora  of  cheese.  If  the  milk  is 
kept  until  it  reaches  a certain  degree  of  acidity,  before  the  rennet  is 
added,  or  if  it  is  allowed  to  curdle  spontaneously,  hundreds  or  thousands 
of  millions  of  bacteria,  mostly  lactic  acid  streptococci,  are  inclosed  in 
every  gram  of  curd.  If  cheese  is  made  by  adding  a home-made  rennet 
infusion  to  sweet  milk,  the  bacteria  in  the  rennet  are  of  prominent  im- 
portance. Such  infusions  contain  usually  about  100  millions  per  cc.,  and 
approximately  1 million  of  rennet  bacteria  is  added  to  each  cubic  centi- 
meter of  milk,  while  commercial  rennet  preparations  in  powder  or  tablet 
form  carry  only  a few  contaminating  organisms.  However,  the  quality 
of  the  microflora  of  the  milk  plays  an  important  role  also  in  those  eases 
where  rennet  infusions  are  used,  and  this  holds  true  especially  in  re- 
gard to  the  udder  bacteria  as  well  as  to  those  added  as  fecal  contamina' 
tions. 

The  influence  of  the  germ  content  of  water,  air,  and  utensils  is  almost 
negligible  insofar  as  numbers  are  concerned,  but  occasionally  organisms 
from  this  source  may  become  responsible  for  certain  abnormal  alterations 
occurring  in  cheese,  and  the  molds  growing  normally  on  and  in  various 
kinds  of  soft  cheese  were  also  originally  derived  from  these  sources,  al- 
though at  present  they  are  frequently  added  as  pure  cultures. 

Pathogenic  organisms  may  sometimes  be  carried  by  soft  cheeses  which 
are  cured  within  a few  days  or  weeks,  while  they  will  die  in  hard  cheeses 
during  the  long  ripening  period.  Milk  pasteurization  will  exclude  any 
danger  from  soft  cheeses,  provided  that  no  disease  carrier  was  allowed 
to  touch  the  products. 

Frequency  of  Microorganisms  in  Cheese. — When  milk  coagulates,  the 
majority  of  its  bacteria,  approximately  70  to  80  per  cent,  are  inclosed 
in  the  curd.  At  this  time  the  germ  content  of  sweet  milk  cheese  is  usually 
lower  than  that  of  sour  milk  cheese,  and  if  the  curd  of  the  sweet  milk 
cheese  is  heated  to  a rather  high  temperature,  as  is  done  with  Swiss  or 
Emmental  cheese,  the  numbers  are  still  further  reduced;  but  soon  a 
rapid  multiplication  sets  in.  After  a few  days  a maximum  is  reached 
in  the  inner  parts,  which  is  followed  by  a persistent  decline,  similar  to 
that  noticeable  in  butter,  while  on  the  surface  a luxuriant  growth  may 
establish  itself,  provided  that  it  is  not  checked  by  a special  treatment  of 
the  rind.  The  following  data  may  serve  as  an  illustration: 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  199 


Millions  per  g. 

Emmental  Cheese 

Cheddar  Cheese 

Sour  Milk  (Harz)  Cheese 

Surface 

Inner  Parts 

Inner  Parts 

Surface 

Inner  Parts 

Fresh  curd 

8 

10 

After  1 day  .... 

450 

30 

70 

70 

6 days 

300 

23 

10  days 

8,500 

62 

41 

13  days 

100 

82 

45  days 

66,000 

55 

10 

150  days 

23,000 

34 

0.5 

The  white  slimy  growth  appearing  upon  the  surface  of  young  sweet  milk 
cheeses  was  found  to  contain  up  to  500,000  millions  of  bacteria  per  g. 

If  the  curing  takes  place  at  low  temperatures,  as  with  American 
Cheddar  cheese,  the  reduction  in  numbers  is  much  slower  than  at  higher 
temperatures,  as  is  shown  by  the  following  counts : 


Millions  per  g 

After  1 Day 

After  15  Days 

After  70  Days 

At  18°  to  20°  C 

523 

145 

4 

At  3°  to  5°  C 

523 

489 

473 

Fig.  48. — Contact  preparations  (X600)  made  from  Gervais  (left)  and  from  Scotch 

Cheddar  cheese  (right). 

The  distribution  of  the  bacteria  is  fairly  uniform  in  young  cheeses, 
but  later  colonies  are  formed  which  act  as  “ripening  centers.”  It  is 
easily  understood  that  reliable  plate  counts  are  hard  to  obtain  from  such 
material,  and  that  direct  microscopic  tests  furnish  much  more  accurate 
and  complete  information.  Figure  48  shows  such  stained  preparations 
made  from  Gervais  cheese,  a French  soft  cheese,  and  from  Scotch 


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Cheddar  cheese.  In  the  first  case,  besides  a few  yeasts  and  rods,  only 
lactic  acid  streptococci  are  visible  in  uniform  distribution,  while  in  the 
second  case  the  characteristic  accumulation  of  lactobacilli  is  very  pro- 
nounced. 

Species  of  Cheese  Organisms—  Lac  tic  acid  bacteria  are  always  most 
abundant  in  sweet  milk  as  well  as  in  sour  milk  cheeses.  Close  to 
the  surface  many  aerobic  lactic  acid  micrococci  are  to  be  found,  while 
streptococci  and  lactobacilli  are  most  numerous  in  the  center,  due  to  their 
tendency  toward  anaerobic  life.  In  soft  cheeses  as  well  as  in  young  hard 
cheeses  streptococci  are  much  more  numerous  than  lactobacilli,  hut  these 
predominate  always  in  ripe  hard  cheeses.  The  Scotch  Cheddar  cheese 
used  for  Fig.  48  was  made  with  a starter  that  contained  nothing  hut 
lactic  acid  streptococci,  yet  this  type  of  lactic  acid  bacteria  was  practi- 
cally absent  in  the  ripe  cheese.  With  American  Cheddar  cheese 
analogous  results  have  been  obtained.1  The  same  change  was  observed  in 
Swiss  cheese  prepared  with  rennet  powder  instead  of  home-made  infus- 
ion. The  percentage  of  both  groups  of  lactic  acid  bacteria  was  found  to 
be  as  follows : 


Percentage 

In  the  Fresh 
Curd 

After  1 Day 

After  17  Days 

Streptococci 

100 

70 

0 

Lactobacilli 

0 

30 

100 

Home-made  rennet  infusions  are  always  rich  in  lactobacilli,  because 
these  predominate  in  the  digestive  tract  of  milk-fed  calves.  On  the  other 
hand,  the  intestinal  lactic  acid  bacteria  (B.  coli  and  aerogenes)  appear- 
ing as  fecal  contaminations  in  cows’  milk  may  become  distinctly  harm- 
ful in  cheese,  because  of  their  ability  to  produce  gases  and  to  exert  an 
unfavorable  influence  upon  taste  and  flavor  of  the  product. 

Other  non-sporulating  and  sporulating  bacteria  are  generally  of  very 
little  importance.  In  a few  cases  they  may  participate  in  the  ripening 
process,  hut  these  are  rather  rare  exceptions. 

Yeasts,  the  habitual  symbionts  of  lactic  acid  bacteria,  are  quite  com- 
mon in  cheeses.  They  are  absent  only  if  the  curd  is  heated  to  55°  C.,  as 
in  making  Swiss  cheese,  because  this  is  their  lethal  temperature. 

Molds  are  of  importance  in  certain  soft  cheeses,  while  they  are  sup- 

1 E.  G.  Hastings,  A.  C.  Evans  and  E.  B.  Hart,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  36, 
1913,  p.  457. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  201 


pressed  in  other  soft  cheeses,  as  well  as  in  all  hard  cheeses  whose  surface 
is  treated  in  a manner  that  prevents  the  development  of  such  fungi. 


2.  BACTERIAL  ACTIVITIES  AND  THE  RIPENING  OF  CHEESE 

If  cheese  is  prepared  aseptic-ally  or  is  made  from  sterilized  curd,  it 
will  show  but  restricted  alterations  which  are  very  different  from  those 
characteristic  of  normal  ripening.  Microorganisms  and  enzymes  are  in- 
dispensable for  obtaining  the  desired  transformations,  which  extend  to  all 
- — nitrogenous  as  well  as  non-nitrogenous — constituents  of  the  cheese.  In 
the  literature,  the  term  ripening  was  repeatedly  used  as  synonymous  with 
metabolism  of  nitrogen  only,  and  many  misunderstandings  have  arisen 
from  the  ambiguous  use  of  that  term.  Undoubtedly,  the  transformation 
of  casein  and  of  other  nitrogenous  compounds  represents  the  most  im- 
portant part  of  the  whole  complex  of  chemical  changes,  but  if  the  curd 
is  freed  from  milk  sugar  by  thorough  washing,  the  decomposition  of 
casein  gives  rise  to  typically  putrid  odors,  although  the  chemical  analysis 
will  furnish  results  not  very  different  from  those  obtainable  with  normal 
cheese.  Therefore,  a correct  insight  into  the  chemical  and  bacteriological 
problems  of  cheese  ripening  is  assured  only  if  comprehensive  investiga- 
tions are  made  of  all  chemical  changes  occurring  in  the  ripening  cheese ; 
besides  the  metabolism  of  casein,  the  transformation  of  milk  sugar  and 
of  fat  are  of  foremost  importance. 

Acid  Formation. — In  hard  cheeses,  from  which  most  of  the  whey  is 
removed  by  pressing,  all  milk  sugar  is  usually  transformed  into  acids 
within  8 to  10  days,  while  in  soft  cheeses  the  higher  wThey  and  sugar 
content  leads  to  a prolongation  of  this  process.  The  large  loaves  of 
hard  cheese  are  soon  free  from  all  oxygen ; accordingly,  the  lactic  acid 
streptococci  and  lactobacilli  find  most  favorable  conditions.  The  in- 
tensity of  acid  formation  in  the  whey,  immediately  before  and  after  the 
cheese  is  formed,  determines  to  a large  extent  the  regular  course  of 
ripening.  Every  type  of  cheese  has  its  characteristic  acidity  curve, 
which  has  been  most  carefully  studied  in  Cheddar,  Swiss,  and  some 
of  the  French  soft  cheeses.  The  total  titratable  acidity  is  equiva- 
lent to  1 to  1 !/2  per  cent  of  lactic  acid  in  hard,  and  to  2 to  4 per  cent 
in  soft  cheeses,  but  because  the  acidity  of  casein  and  of  acid  phosphates 
influences  the  results  so  obtained,  these  figures  are  only  of  relative 
value,  and  do  not  represent  the  real  amount  of  lactic  acid  present  in 
the  cheese.  Especially  in  old  hard  cheeses  frequently  all  free  acid 
is  gone,  as  is  shown  by  determinations  of  their  hydrogen-ion  concen- 
tration. 

If  the  acid  formation  is  too  weak,  the  casein  decomposition  becomes 


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too  intensive,  and  gas  producing  bacteria  may  become  very  active,  while 
an  abnormally  strong  acid  formation  will  suppress  the  desired  nitrogen 
metabolism,  and  no  typical  ripening  will  take  place. 

Transformation  of  Lactic  Acid. — The  free  acid  first  formed  is  more 
or  less  completely  neutralized  by  casein,  calcium,  and  the  ammonia 
liberated  in  the  decomposition  of  the  organic  nitrogen  compounds,  and 
the  lactates  are  further  changed  to  volatile  acids  and  carbon  dioxide. 
The  reduction  in  acidity  has  been  ascertained  by  numerous  titrations, 
but  a more  accurate  insight  will  be  gained  if  these  experiments  are 
repeated  and  the  hydrogen-ion  concentrations  determined.  For  ex- 
ample, the  titratable  acidity,  calculated  as  lactic  acid,  showed  the 
following  behavior  in  Limburger  cheese : 

Ripe  Cheese 

Young  Cheese  Inner  Parts  Surface 

3.21  per  cent  1.35  per  cent  0.89  per  cent 

Probably  the  hydrogen  number  would  have  shown  that  no  acidity 
remained  in  the  surface  part  of  the  cheese.  The  numerous  micro- 
organisms living  in  this  layer  produce  much  ammonia  for  neutraliza- 
tion, and  they  oxidize  the  lactates,  so  that  an  alkaline  reaction  soon 
results. 

Formic,  acetic,  and  propionic  acids  are  very  common  products  of 
the  transformation  of  lactates.  Swiss  cheese  is  exceptionally  rich  in 
propionic  acid ; the  bacteria  active  in  this  case  are  of  great  influence 
upon  the  texture  and  flavor  of  first-class  Emmental  cheese.  Part  of 
the  volatile  acids  forms  esters  with  the  small  quantities  of  ethyl  alcohol, 
produced  simultaneously  by  different  bacteria  and  by  yeasts,  if  these 
are  present;  such  esters  are  also  of  considerable  influence  upon  taste 
and  flavor  of  the  cheese. 

Decomposition  of  Casein. — That  the  decomposition  of  casein  pro- 
ceeds rather  differently  in  hard  and  in  soft  cheeses  is  evidenced  by  a 
superficial  examination  of  the  various  kinds,  after  they  have  well 
ripened.  The  texture  of  the  curd  shows  that  a greater  percentage  of 
casein  is  changed  to  soluble  compounds  in  the  soft  cheeses,  which 
when  overripe  may  even  assume  a semi-liquid  consistency.  Their 
stronger  flavor  indicates  at  the  same  time  that  more  ammonia  is  pro- 
duced than  in  hard  cheeses.  In  the  latter  only  x/3  to  x/2  of  the  total 
nitrogen  is  found  in  the  form  of  soluble  compounds,  and  these  are 
present  chiefly  in  the  form  of  certain  amino  acids,  which  have  proved 
to  be  of  great  influence  upon  the  characteristic  taste  of  these  cheeses. 
If  the  decomposition  of  the  casein  keeps  within  narrow  limits,  as  in 
Gervais  and  in  Edam  cheese,  the  low  figures  for  soluble  and  for  amino 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  203 


nitrogen  demonstrate  this  just  as  clearly  as  do  the  mild  tastes  and 
flavors  of  these  products.  On  the  other  hand,  a high  percentage  of 
soluble  and  of  ammonia  nitrogen  is  characteristic  of  such  cheeses  as 
Limburger  and  Camembert,  while  a comparatively  large  percentage  of 
amino  nitrogen  is  indicative  of  the  high  quality  of  Emmental,  Cheddar, 
Roquefort,  Stilton,  and  similar  cheeses.  The  following  data  gathered 
from  analyses  of  ripe  cheeses  may  illustrate  these  relations : 


Per  Cent  of  Soluble  N 

Kind  of  Cheese 

Taste  and  Flavor 

Per  Cent  of  Total  N 
Soluble 

Amino-N 

Ammonia-N 

Gervais 1 

Edam / 

Very  mild 

22 

27 

20 

11 

6 

2 

Emmental 1 

Mild 

33 

52 

7 

Swedish  hard  cheese  . / 

38 

00 

7 

Cheddar 1 

Stronger 

50 

55 

7 

Roquefort / 

52 

45 

9 

Camembert 1 

Very  strong 

100 

15 

10 

Limburger / 

99 

5 

12 

These  nitrogen  figures  vary  greatly  in  each  cheese  with  its  age, 
and  smaller  variations  are  also  noticeable  in  different  samples  of  ripe 
cheeses  of  the  same  type.  A fundamental  difference  exists  between 
hard  and  soft  cheeses  insofar  as  in  the  first  case  the  transformation 
of  the  casein  proceeds  uniformly  throughout  the  whole  curd,  while 
in  the  second  case  the  changes  start  at  the  surface  and  the  white  sour 
center  remains  unchanged  for  a long  time.  Here  again  it  is  demon- 
strated that  anaerobic  processes  predominate  in  the  hard,  and  aerobic 
ones  in  the  soft  cheeses.  Naturally,  taste  and  flavor  are  dependent 
not  only  on  the  transformation  of  casein,  but  also  on  the  presence 
of  other  substances,  especially  of  products  of  the  decomposition  of  fat, 
and  of  salt.  These  substances,  however,  are  to  be  found  also  in  bard 
cheeses  more  frequently  close  to  the  surface,  and  because  of  this  fact 
some  authors  have  drawn  the  incorrect  conclusion  that  aerobic  or- 
ganisms were  of  equal  importance  in  hard  as  well  as  in  soft  cheeses. 

Enzymatic  and  Bacterial  Action  upon  Casein. — The  ripening  of  sour 
milk  cheeses  shows  most  distinctly  that  microorganisms  and  their 
enzymes  are  of  fundamental  importance.  Cheese  prepared  aseptically 
does  not  ripen  at  all,  as  was  first  demonstrated  by  Duclaux  in  France 
about  fifty  years  ago.  If  rennet  is  used,  the  pepsin  contained  therein 


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participates  in  peptonizing  the  casein,  provided  the  acidity  is  suffi- 
ciently high  (above  0.3  per  cent  lactic  acid),  as  is  the  case  m Cheddar 
and  most  soft  cheeses. 

Peptonizing  microorganisms  and  their  enzymes  are  present  in  every 
milk ; most  important  among  them  are  the  micrococci  drawn  from  the 
udder.  They  become  active  in  the  cheese  as  soon  as  the  lactic  acid 
streptococci  have  produced  enough  acid.  At  the  same  time  the  acid 
produced  activates  the  rennet  pepsin,  and  the  rennet  in  its  turn  stimu- 
lates the  otherwise  weak  proteolytic  actions  of  the  streptococci,  as  has 
been  discovered  recently.1  The  formation  of  the  relatively  large  quan- 
tities of  amino  acids  present  in  hard  cheeses,  is  largely  the  work  of 
lactobacilli,  while  in  soft  cheeses  various  fungi,  molds  as  well  as  yeasts, 
are  most  active  in  the  dissolution  and  transformation  of  the  casein. 
Symbiotic  and  antagonistic  actions  between  different  groups  of  micro- 
organisms and  their  enzymes  are  of  great  importance  in  these  com- 
plicated processes.  Every  type  of  cheese  shows  its  own  peculiarities, 
and  it  is  easily  understood  that  the  investigations  made  during  the  last 
twenty  or  thirty  years  have  by  no  means  solved  all  the  problems,  although 
in  general  the  whole  situation  is  now  well  cleared.  A Swiss  bacteriologist, 
E.  von  Freudenreieh  of  Bern,  has  made  most  valuable  contributions  in 
this  respect.  He  has  especially  shown  that  the  high  quality  of  hard 
cheeses  depends  mostly  on  the  work  of  lactobacilli,  while  aerobic  sporu- 
lating  bacilli  are  of  no,  or  of  subordinate,  importance,  although  E. 
Duclaux  had  thought  them  to  be  most  active  and  had  called  them,  there- 
fore, Tyrothrix,  that  is,  cheese-thread.  Strictly  anaerobic  spore-formers 
are,  as  a rule,  equally  unimportant ; but  they  are  very  active  in  a few 
little  known  European  cheeses  of  inferior  quality,  and  sometimes  they 
cause  undesirable  changes  in  other  cheeses. 

Microflora  of  Different  Kinds  of  Cheese. — Peptonizing  micrococci 
and  lactic  acid  streptococci  are  present  in  every  cheese,  while  the  other 
microorganisms  participating  in  the  transformation  of  the  casein  differ 
greatly  with  the  various  types  of  cheese. 

Hard  cheeses  of  fine  taste  and  flavor,  such  as  Cheddar,  Edam, 
Emmental  or  Swiss  cheese,  and  Swedish  hard  cheese,  are  all  rich  in 
lactobacilli. 

The  various  cheeses  of  Roquefort  type,  such  as  Stilton,  Wensleydale, 
blue-Dorset,  and  Gorgonzola,  contain  also  numerous  lactobacilli,  but 
the  growth  of  a bluish-greenish  mold,  Penicillium  Roqueforti,  within 
the  curd  is  most  characteristic.  This  fungus  is  added  in  the  form  of 
moldy  bread  when  Roquefort  cheese  is  formed.  The  ripening  cheese 

1 Chr.  Barthei.  und  E.  Sandberg,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  49,  1919,  p.  392. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  205 


is  pricked  with  needle-like  instruments  in  order  to  assure  a sufficient 
growth  of  the  aerobic  fungus  in  the  inner  parts  of  the  loaves. 

The  flat  soft  cheeses,  such  as  Brie  and  Camembert,  are  covered  by 
different  molds;  white  Penieillium-species  and  varieties  of  Oidium  lac- 
tis  are  most  numerous  among  them.  The  gradual  advance  of  their 
proteolytic  enzymes  from  the  surface  toward  the  center  can  easily  be 
ascertained  by  examining  cuts  of  cheeses  of  different  age.  Aerobic 
bacteria  take  part  in  this  process;  upon  ripe  cheeses  they  appear  as  a 
characteristic  reddish  coating.  If  it  is  customary  to  use  such  soft 
cheeses  while  they  are  very  young,  as  is  done,  for  example,  with 
Gervais  and  Neufchatel  cheese,  the  mold  growth  does  not  become  very 
conspicuous,  although  its  effect  is  quite  noticeable  if  these  cheeses  are 
kept  for  a few  days. 

In  another  type  of  soft  cheeses  the  mold  growth  is  artificially  sup- 
pressed by  a special  treatment,  which  only  permits  the  development 
of  certain  proteolytic  bacteria  upon  the  surface,  where  they  grow  in  a 
thick  slimy  layer.  Their  metabolic  products  are  partly  the  same  as  in 
typical  putrefaction  (putresc-in,  cadaverin,  indol,  and  hydrogen  sul- 
fide) ; accordingly,  much  depends  on  the  personal  disposition  whether 
or  not  such  a cheese,  like  Limburger,  is  accepted  as  human  food.  Very 
interesting  data  have  been  obtained  concerning  the  symbiotic  action  of 
the  bacteria  growing  in  this  cheese,  which  clearly  demonstrate  that 
results  may  be  rather  misleading  if  pure  cultures  only  are  tested.  When 
the  two  species  which  are  most  characteristic  of  this  cheese,  were  grown 
in  milk,  separately  or  combined,  the  following  changes  in  the  distribu- 
tion of  nitrogen  were  observed : 


Increase  or  Decrease  in  Per  Cent 
of  Total  Nitrogen 

Soluble 

Nitrogen 

Amino 

Nitrogen 

Ammonia 

Nitrogen 

Pure  culture  of  B.  casei  limburgensis 

+ 1.26 

- 1.50 

+ 1.13 

Pure  culture  of  M . casei  liquefaciens 

+51.06 

+ 6.06- 

+ 1.13 

Mixed  culture  of  both  organisms 

+78.28 

+31.88 

+ 9.30 

Transformation  of  Fat. — Much  greater  differences  in  regard  to  the 
transformation  of  fat  than  in  the  transformation  of  milk  sugar  and  of 
casein  are  noticeable  with  the  various  types  of  cheeses.  Since  cheeses  are 
made  from  cream,  from  whole  milk,  or  from  skim  milk,  their  original 
fat  content  may  vary  widely.  Furthermore,  in  certain  cases  the 
growth  of  aerobic  organisms,  especially  of  strongly  fat-splitting  molds, 
is  favored  by  the  form  of  the  cheeses  and  the  technique  applied,  as 
in  Brie,  Camembert,  and  Roquefort  cheeses,  while  in  other  cases  prac- 


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tically  all  surface  growth  is  suppressed  by  rubbing  the  rind  with  salt 
or  by  covering  it  with  paraffin.  Because  the  splitting  of  fat  is  almost 
exclusively  the  work  of  aerobic  bacteria  and  fungi,  it  proceeds  most 
vigorously  in  soft  cheeses  of  high  fat  content.  If  such  cheeses  become 
too  ripe,  their  flavor  turns  distinctly  rancid,  as  may  be  noticed  especially 
with  Roquefort  cheese  during  summer.  In  hard  cheeses  more  of  the 
liberated  fatty  acids  are  found  close  to  the  surface,  while  flat  soft 
cheeses  may  contain  more  of  them  in  the  center,  due  to  evaporation 
and  oxidation  at  the  surface.  The  following  figures  illustrate  these 
differences,  giving  the  amounts  of  acids  in  gram  per  1000  g.  cheese : 


Butyric  Acid 

Caproic  Acid 

f outer  parts  

1 232 

0 928 

Emmental  cheese  \ . 

1 inner  parts 

f outer  parts 

0.176 

0.466 

0.116 

0.128 

Brie  cheese  < . 

{ inner  parts 

0.572 

0.139 

The  fatty  acids  are  partly  neutralized  by  ammonia.  The  glycerol 
is  rapidly  transformed,  as  is  the  case  in  rancid  butter.  Little  fat  is 
formed  from  casein  by  molds,  but  contrary  to  an  opinion  held  for 
some  time  by  different  authors  this  process  plays  no  important  part 
in  ripening  cheese. 

Taste  and  Flavor  of  Cheese. — Not  all  of  the  substances  tvhich  in 
their  entirety  are  responsible  for  the  specific  taste  and  flavor  of  the  dif- 
ferent kinds  of  cheese  are  known  at  present,  but  it  is  beyond  dispute 
that  all  of  them  are  regular  products  of  the  transformation  of  milk 
sugar,  casein,  fat,  and  of  their  derivatives.  Formerly,  much  interest 
centered  upon  the  discovery  of  aroma  producing  organisms  in  cheese 
as  well  as  in  butter,  and  it  is,  indeed,  not  very  difficult  to  isolate  from 
cheese  different  strains  of  bacteria  and  fungi  which  are  characterized 
by  their  abilities  to  produce  peculiar  flavors,  even  when  grown  on  the 
substrates  commonly  used  in  the  laboratory.  However,  these  char- 
acters are  very  easily  lost ; they  are  not  species  marks,  but  merely 
peculiarities  temporarily  acquired  under  the  influence  of  prevailing 
environmental  conditions. 

The  refreshing  taste  of  young  cheeses,  such  as  Neufchatel  or 
Gervais,  is  mostly  due  to  lactic  acid,  acetic  acid,  acetone,  alcohol,  and 
various  esters.  The  taste  of  hard  cheeses,  such  as  Cheddar  and  Em- 
mental  cheese,  is  largely  dependent  upon  the  quantities  of  alanin, 
glycin,  and  other  amino  acids  present.  Certain  strains  of  lactic  acid 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  207 


and  of  propionic  acid  bacteria  have  been  found  to  act  favorably 
upon  the  flavors  of  these  cheeses.  Taste  and  flavor  of  Roquefort  and 
of  similar  cheeses  are  greatly  influenced  by  substances  produced  by 
Penicillium  Roqueforti  in  the  transformation  of  fats.  Free  fatty  acids 
contribute  to  the  pungent  flavor  of  ripe  soft  cheeses,  and  the  ammonium 
salts  of  caproic,  caprylic  and  caprinic  acids  are  in  the  first  place 
responsible  for  the  peculiar  “cheesy”  odor  of  such  products. 

Texture  of  Cheese ; Eye  Formation. — Preparation  and  manipulation 
of  the  curd,  forming,  pressing,  and  further  treatment  of  the  young 
cheeses  are  largely  responsible  for  the  initial  differences  in  the  struc- 
ture, which  in  their  turn  regulate  the  chemical  processes  that  lead  to 
more  or  less  far-reaching  changes  of  the  texture  in  the  ripening  cheese. 
In  soft  cheeses  the  white  loose  acid  curd  is  gradually  transformed 
into  a smooth,  gelatinous,  semi-transparent  paste,  while  in  hard  cheeses 
these  alterations  are  much  less  pronounced,  due  to  the  restricted  acid 
formation  and  the  less  intense  dissolving  of  the  casein. 

The  firmer  and  more  plastic  texture  of  the  hard  cheeses  favors  the 
appearance  of  uniformly  distributed  openings  in  the  curd,  usually 
called  eyes.  Gases,  mostly  carbon  dioxide,  liberated  in  the  fermentative 
changes  of  lactates  and  of  glycerol,  accumulate  at  spots  where  drops 
of  whey  remained  inclosed  in  the  curd.  This  eye  formation  is  especially 
characteristic  in  Emmental  cheese,  and  no  cheese  of  this  type  is  accepted 
as  of  first  quality,  if  it  is  not  “well  opened,”  that  is,  if  it  does  not 
have  a sufficient  number  of  very  regularly  formed  and  uniformly  dis- 
tributed eyes,  each  of  about  1 cm.  diameter.  The  gases  which  form  these 
eyes  are  liberated  in  tbe  transformation  of  lactates  to  propionates. 
Because  this  process  is  of  minor  importance,  it  is  easily  understood 
why  occasionally  “blind”  Emmental  cheeses  are  found  which  are  also 
of  good  taste  and  flavor.  In  the  manufacture  of  other  hard  cheeses 
much  less  attention  is  paid  to  this  eye  formation;  in  fact,  it  is  almost 
entirely  absent  in  many  cases.  The  normal  “opening”  of  the  cheeses 
should  not  be  confounded  with  “gassiness,”  that  is  an  abnormal  and 
excessive  production  of  gas  by  organisms  of  fecal  origin. 

Abnormal  Alterations. — If  the  transformations  of  milk  sugar,  of 
casein,  and  of  fat  do  not  proceed  simultaneously  in  a well  balanced 
manner  as  in  normal  cheese,  the  result  will  be  unsatisfactory  in  one 
or  another  direction.  Excessive  acid  formation  makes  an  unripe  sour 
product ; predominance  of  casein  decomposition  causes  a disagreeable 
bitter  taste  and  putrid  odor;  too  far-reaching  splitting  of  the  fats 
gives  a distinctly  rancid  cheese.  However,  abnormal  tastes  and  flavors 
may  also  be  produced  by  certain  strains  of  microorganisms  which  do 
not  belong  to  the  regular  microflora  of  cheese.  Yeasts  have  repeatedly 


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been  found  to  act  unfavorably,  especially  in  Cheddar  cheese,  among  the 
bacteria  those  of  fecal  origin  are  least  desirable.  Cheese  poisoning 
is  partly  due  to  the  presence  of  large  numbers  of  such  organisms  (P>. 
coli  and  its  relatives) , but  B.  botulinus  has  also  been  found  occasionally. 
As  said  before,  overripe  cheeses  contain  substances  of  typically  putrid 
character;  it  is  very  probable  that  in  many  cases  where  toxic  effects 
have  been  observed,  such  cheese  had  been  eaten. 

Abnormal  milk,  such  as  colostrum,  or  as  is  obtained  toward  the 
end  of  lactation,  is  not  suited  for  the  manufacture  of  high  grade  cheese. 
The  regular  examination  of  milk  and  of  rennet  in  the  fermentation 
test  (see  Plate  X)  is  the  most  effective  means  of  discovering  faulty 
material  that  might  cause  abnormal  alterations  in  the  cheese.  The 


Fig.  49.- — Blown  Swiss  cheese  (TA  nat.  size). 

formation  of  a smooth  curd  with  none  or  only  a few  holes  promises 
a satisfactory  outcome,  while  a blown  or  torn  curd  serves  as  a warning 
that  failures  will  result,  unless  they  are  promptly  checked  by  the 
application  of  preventive  methods. 

Gassiness;  Blown  Cheese. — Excessive  gas  formation  occurs  if  milk 
from  inflamed  udders  is  used,  or  if  milk  or  rennet  are  heavily  con- 
taminated by  fecal  organisms  (B.  coli  and  aerogenes,  or  B.  arnylo- 
bacter).  Not  all  mastitis  streptococci,  but  some  of  them,  produce  con- 
siderable quantities  of  carbon  dioxide  from  milk  sugar,  and  mastitis 
milk  itself  is  usually  changed  to  such  an  extent  in  its  chemical  com- 
position that  it  disturbs  the  regular  course  of  cheese  ripening.  B.  coli 
and  aerogenes  liberate  large  quantities  of  carbon  dioxide  and  of  hydro- 
gen from  lactose  during  the  first  few  days,  sometimes  even  while  the 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  209 


cheese  is  still  in  the  press.  Fig.  49  shows  a blown  Swiss  cheese  that 
was  cracked  by  the  excessive  gas  formation.  The  discoloration  of  its 
torn  center  indicates  that  other  unfavorable  alterations  occur  in  such 
cheeses.  In  fact,  it  is  more  on  account  of  the  disagreeable  taste  and 
flavor  of  blown  cheeses  than  because  of  the  change  in  texture  that 
their  market  value  is  very  much  reduced.  If  anaerobic  bacilli  (B. 
amylobacter)  get  into  the  cheese  in  large  numbers,  the  gas  formation 
does  not  become  noticeable  before  several  weeks  have  elapsed,  because 
the  carbon  dioxide  and  hydrogen  liberated  by  them  is  derived  from  the 
lactates  which  they  transform  into  butyrates.  The  latter  again  act 
unfavorably  upon  taste  and  flavor.  Occasionally  yeasts  are  responsible 
for  gassy  cheese,  but  in  general  they  are  much  less  detrimental  than 
the  two  groups  of  bacteria  named. 

Blown  cheese  is  not  always  characterized  by  very  large  and  irregular 
holes.  Sometimes  the  whole  cheese  becomes  rather  spongy,  because 
the  gases  have  made  innumerable  small  holes,  each  of  about  2 to  5 
mm.  diameter.  Large  holes  are  formed  if  the  gas  producing  organisms 
have  entered  the  curd  as  colonies  attached  to  dirt  particles,  while  small 
holes  are  the  result  of  a more  even  distribution  of  single  cells  or  of 
small  conglomerates,  as  is  regularly  the  case  in  milk  that  has  passed 
through  the  centrifuge. 

Abnormal  Texture  and  Color  of  Cheese. — The  disturbances  in  the 
structure  of  cheese  caused  by  excessive  gas  formation  leads,  as  a rule, 
to  further  deteriorations  in  its  texture.  Parts  of  the  curd  become 
abnormally  hard,  while  others  remain  too  soft  and  become  more  or 
less  slimy.  Similar  alterations  may  be  due  to  the  presence  of  slime 
producing  organisms,  whose  activity,  however,  is  usually  confined  to 
the  surface,  where  they  are  less  detrimental  than  they  would  be,  if 
they  would  also  attack  the  inner  parts. 

Abnormal  pigmentation  of  cheeses  is  only  partly  caused  by  micro- 
organisms. If  sour  or  well  ripened  milk  is  used,  frequently  small 
amounts  of  metals  are  transferred  from  the  utensils  to  the  cheese,  where 
they  are  changed  to  sulfides  which  make  the  curd  bluish,  greenish,  gray 
or  black.  If  the  cheeses  are  kept  on  boards  made  from  spruce,  the  outer 
parts  show  often  a reddish-brownish  coloration  which  is  caused  by  sub- 
stances entering  the  cheeses  from  the  wooden  supports. 

In  other  cases  bacteria  and  fungi  are  the  causes  of  abnormal  yellow, 
greenish,  bluish,  reddish,  brown,  or  black  spots  occurring  upon  or  within 
the  cheeses.  Contrary  to  the  diffused  staining  of  non-bacterial  origin, 
the  pigment  production  by  bacterial  or  fungous  colonies  is  sharply  local- 
ized. With  proper  technique,  no  great  harm  is  to  be  expected  from  most 
of  these  organisms ; only  certain  red,  brown,  and  black  varieties  of  lactic 


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acid  and  propionic  acid  bacteria  may  become  rather  troublesome,  because 
as  kinsmen  of  typical  cheese  bacteria,  they  find  most  suitable  environ- 
mental conditions  in  every  normal  cheese.  Accordingly,  they  must  be 
traced  to  their  source,  and  the  initial  infection  must  be  prevented. 

3.  MEANS  OF  REGULATING  THE  ACTIVITY  OF  MICROORGANISMS  IN 

CHEESE 

The  qualities  of  the  different  types  of  cheese  are  mainly  influenced 
by  the  qualities  of  milk  and  of  rennet,  and  by  the  technique  applied  in 
each  case.  It  is  very  interesting  to  note  how  the  bacteriological  investiga- 
tions made  during  the  last  decades  have  elucidated  many  of  the  methods, 
employed  by  experienced  cheese  makers,  in  regard  to  their  inherent 
reasons,  and  that  now  the  various  technical  modifications  can  be  chosen 
and  applied  much  more  intelligently  and  successfully  than  at  the  time 
when  all  depended  on  experience  and  practical  skill.  The  use  of  pas- 
teurized milk  and  of  pure  culture  starters  has  further  stabilized  the 
cheese  industry,  although  much  remains  to  be  done  in  this  respect  until 
the  whole  problem  will  have  been  brought  to  an  equally  satisfactory 
status  as  has  been  reached  in  regard  to  cream  ripening  and  butter 
making. 

Influence  of  Milk  and  of  Rennet. — The  quality  of  the  milk  used  is  of 
importance  chemically  as  well  as  biologically.  Changes  in  its  lactose 
content  may  lead  to  abnormally  weak  or  abnormally  strong  acid  forma- 
tion with  its  harmful  consequences.  An  unusually  high  fat  content 
invites  rancidity  if  soft  cheeses  are  made.  Very  clean  milk  can  be  used 
successfully  only  if  good  starters  are  available,  otherwise  no  clean  and 
vigorous  acid  formation  would  be  assured.  The  fermentation  test  will 
furnish  the  necessary  information ; a smooth  curd  with  very  little  whey 
of  clean  taste  and  flavor  is  most  desirable  (see  Plate  X,  Fig.  lb). 

Rennet  likewise  acts  chemically  as  well  as  biologically.  Because  of 
the  pepsin  contained  therein  a relatively  large  amount  of  rennet  hastens 
the  ripening  process,  at  least  in  all  cheeses  of  sufficiently  high  acidity. 
Rennet  powders  or  tablets  do  not  contain  a valuable  microflora ; in  con- 
nection with  pasteurized  milk  normal  cheese  can  be  obtained  only  if  good 
starters  are  used.  In  home-made  rennet  infusions,  however,  the  useful 
lactobacilli  are  very  numerous,  provided  that  the  stomachs  have  been 
carefully  cleaned.  If  this  was  not  done,  or  if  the  rennet  infusion  has 
been  contaminated  from  other  sources,  gas  producing  organisms  may 
predominate.  Again  the  fermentation  test  will  render  very  valuable 
service  in  the  control  of  the  rennet,  together  with  determinations  of  its 
acidity. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  211 


Influence  of  Technique. — The  extent  to  which  the  milk  is  ripened, 
that  is,  held  for  spontaneous  souring,  before  it  is  coagulated,  the  curdling 
and  the  manner  in  which  the  curd  is  manipulated,  determine  in  the 
first  place  the  kind  of  microflora  and  its  activity  in  the  young  cheese. 
If  the  curd  is  heated,  it  becomes  harder;  and  the  higher  the  temperature, 
the  more  organisms  are  eliminated,  excepting  the  lactobacilli  that  do  not 
suffer  even  if  55°  C.  are  applied,  as  in  the  making  of  Swiss  cheese. 
Yeasts  are  almost  completely  killed  by  this  treatment,  otherwise  they 
might  act  very  unfavorably  as  gas  producers. 

If  the  cheeses  are  pressed,  their  whey  content  is  more  or  less  reduced ; 
accordingly,  the  curd  becomes  firmer,  and  the  acid  formation  is  reduced. 
It  depends  on  the  form  and  the  size  of  the  loaves  whether  the 
ripening  will  be  more  under  the  influence  of  aerobic  or  of  anaerobic 
organisms,  as  was  discussed  before.  T emperature  and  humidity  of  the 
curing  and  storage  rooms  affect  the  water  content  of  the  cheeses,  and 
foster  or  check  the  development  and  activities  of  the  various  groups  of 
microorganisms  and  of  their  enzymes.  With  Roquefort  and  American 
Cheddar  cheeses  very  good  results  are  obtained  at  comparatively  very 
low  temperature.  Little  harm  can  be  done  by  the  gas  producing  bacteria 
below  10°  C. 

The  salting  of  the  cheese  acts  differently  according  to  its  applica- 
tion. If  the  salt  is  added  to  the  curd,  but  not  evenly  mixed  with  it,  it 
will  retard  the  acid  formation  in  places ; the  dissolution  of  the  casein 
may  then  prevail  to  such  an  extent  that  soft  putrid  spots  will  appear. 
If  the  salt  is  applied  from  the  outside,  it  becomes  possible  to  use  this 
means  for  stimulating  or  retarding  the  biochemical  transformations 
within  the  cheese.  Because  more  salt  accumulates  close  to  the  rind,  it 
checks  the  action  of  susceptible  organisms  in  this  zone,  as  for  instance 
that  of  the  propionic  acid  bacteria  in  Swiss  cheese;  the  characteristic 
eyes  are  absent  within  a few  inches  from  the  rind  even  in  otherwise 
well  opened  cheeses.  The  bathing  of  Limburger  in  brine  and  the  so-called 
“smearing”  of  its  surface,  which  is  kept  moist  continually,  suppresses 
almost  completely  the  growth  of  molds  that  is  not  desired  in  this  cheese. 

Special  treatment  of  the  rind  helps  to  favor  or  to  exclude  aerobic 
organisms.  Most  effective  in  checking  such  growth  is  the  application 
of  a paraffin  coating,  as  is  now  widely  adopted  for  American  Cheddar 
and  for  Swedish  hard  cheeses.  It  has  proved  equally  useful  for  Edam 
and  Roquefort  cheeses,  provided  that  in  the  latter  ease  the  inner  parts 
are  properly  aerated  by  pricking.  Placing  the  cheeses  in  hay  or  straw 
serves,  on  the  other  hand,  as  a rather  crude  inoculation  of  the  rind  with 
molds  and  other  aerobic  organisms. 

In  regard  to  abnormal  alterations  hardly  anything  can  be  done  after 


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the  cheese  is  made,  except  that  by  cooling  (packing  in  ice)  excessive  gas 
formation  can  be  checked.  In  all  other  respects  “prevention  is  better 
than  cure,”  and  the  fermentation  tests  permit  an  accurate  control  of 
milk,  rennet,  and  water.  Adding  nitrate  to  milk  (20  to  60  g.  per  25 
gallons)  reduces  to  some  extent  the  danger  of  getting  blown  cheeses. 
B.  coli  and  aerogenes  acquire  the  oxygen  they  need,  first  from  the  nitrate, 
which  is  transformed  to  ammonia;  in  the  meanwhile  the  milk  sugar  is 
changed  to  lactic  acid  and  no  longer  available  to  them.  Butyric 
acid  bacteria  and  yeasts,  however,  are  not  hindered  by  the  addition  of 
nitrate.  Occasionally  the  saltpeter  itself  was  found  heavily  infested  by 
yeasts,  and  its  use  proved  distinctly  harmful. 

If  different  kinds  of  cheeses  are  made  side  by  side,  or  if  the  manu- 
facture of  one  kind  is  replaced  by  that  of  another,  it  may  happen  that 
queer  “crosses”  are  produced,  as  for  instance  Camembert  with  Lim- 
burger  flavor.  Local  varieties,  as  are  characteristic  of  the  one  type  of 
cheese,  are  liable  to  continue  their  growth  and  activity  for  a while  in 
the  other  cheese  despite  the  changed  technique.  Therefore,  great  care 
is  necessary  if  cheeses  of  different  kind  are  to  be  made  simultaneously. 
Separate  sets  of  utensils  and  separate  rooms  are  necessary  for  obtaining 
faultless  products.  General  disinfection  and  introduction  of  the  desired 
organisms  must  accompany  the  change  from  one  to  another  type  of 
cheese  making. 

Use  of  Pasteurized  Milk  and  of  Starters. — It  would  be  best  if  for 
cheese  making  as  for  butter  making  pasteurized  milk  and  selected 
starters  could  be  used  generally.  At  present,  however,  this  goal  has 
not  been  reached.  With  soft  cheeses  the  least  difficulties  are  encoun- 
tered. Heated  milk  gives  a soft  curd  which  offers  no  disadvantage 
in  such  cases,  and  the  active  microorganisms  are  well  known,  therefore, 
first-class  starters  can  be  prepared.  Hard  cheeses  offer  greater  diffi- 
culties, mainly  because  of  the  abnormal  consistency  of  the  curd.  That 
excellent  hard  cheeses  can  be  made  from  milk  pasteurized  at  high 
temperatures,  was  first  proven  by  Danish  and  Swedish  cheese-makers. 
But  because  the  chances  are  very  slight  that  pathogenic  organisms  are 
carried  by  hard  cheeses,  the  use  of  clean  raw  milk  is  equally  satisfactory 
or  even  preferable,  because  of  the  firmer  texture  of  the  curd.  This 
point  is  of  greatest  importance  in  the  manufacture  of  Swiss  cheese. 

The  low  germ  content  of  pasteurized  and  of  clean  raw  milk  necessitates, 
of  course,  the  use  of  starters.  Sour  milk,  buttermilk,  and  whey  have 
served  as  such  for  a considerable  length  of  time.  Carefully  prepared 
rennet  infusions  contain  all  the  bacteria  needed  for  Swiss  and  other 
hard  cheeses.  Moldy  bread  has  long  been  used  for  inoculating  Roque- 
fort cheese ; in  Stilton  cheese  the  fungi  were  transplanted  by  inserting 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  CHEESE  213 

plugs  taken  from  old  cheeses.  Quite  generally  a heavy  inoculation  of 
the  milk  with  the  desired  microorganisms  proves  very  useful ; it  can 
easily  be  made  by  adding  to  the  milk  an  emulsion  prepared  from  a 
piece  of  first-grade  half  ripe  cheese  of  the  type  to  be  made. 

Pure  Cultures  for  Cheese  Ripening. — Extensive  and  successful  tests 
with  selected  pure  cultures  have  first  been  made  in  the  manufacture 
of  Swiss  cheese  by  E.  von  Freudenreich  at  the  Experimental  Station 
Bern-Liebefeld,  Switzerland.  Lactobacilli  of  different  types  have  been 


Fig.  50. — French,  Swiss  and  Italian  cultures  for  cheese  ripening  (|-  nat.  size). 

used ; one  called  B.  casei  e was  found  to  be  most  useful,  especially  when 
combined  with  pure  cultures  of  propionic  acid  bacteria.  Such  cultures 
are  now  being  supplied  by  various  institutions  in  Europe  ; in  the  United 
States  the  Dairy  Division  of  the  Federal  Department  of  Agriculture 
has  made  successful  experiments,  and  cultures  may  be  obtained  from 
there. 

For  Cheddar  cheese  pure  cultures  of  various  types  of  lactic  acid 
streptococci  as  well  as  of  lactobacilli  are  used;  the  latter  give  a more 
highly  flavored  product,  as  was  to  be  expected.1 

1 W.  Stevenson,  Trans.  Highland  and  Auric.  Society  of  Scotland,  5.  Ser.r  vol.  30, 
1918,  pp.  97-125, 


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For  the  various  kinds  of  French  soft  cheeses,  such  as  Brie,  Camem- 
bert,  Coulommiers,  and  Roquefort,  selected  cultures  are  now  being 
supplied  in  liquid  and  in  powder  form  by  the  Institut  Pasteur  of  Paris 
and  by  several  commercial  firms. 

In  Italy  an  “ Associazione  pro  Grana”  furnishes  cultures  for  the 
hard  Italian  cheese,  so-called  Parmesan  or  Grana. 

Some  of  the  cultures  as  they  are  supplied  by  experimental  stations 
and  by  the  trade,  are  shown  in  Fig.  50. 


CHAPTER  XII 


SEWAGE  DISPOSAL 

A never  failing  supply  of  clean  water  is  of  fundamental  importance 
on  the  farm.  The  quality  of  milk  and  of  dairy  products  depends  very 
much  on  the  purity  of  the  water,  as  was  repeatedly  pointed  out  on 
the  preceding  pages,  and  the  same  holds  true  in  regard  to  the  general 
health  of  man  and  animal.  Unfortunately,  pollution  of  well  and  spring 
water  is  by  no  means  rare.  Carelessness  in  disposing  of  the  discharges 
from  the  farm  is  much  too  wide-spread.  Wherever  running  water  is 
available  it  is  frequently  used  to  carry  the  wastes  away,  often  without 
giving  adequate  consideration  to  the  inconvenience  and  possible  danger 
such  practice  may  involve.  Where  running  water  is  scarce,  careless 
disposal  of  human  excrements  and  of  other  waste  products  does  not 
only  constitute  a serious  nuisance,  but  again  it  may  become  dangerous, 
because  at  such  places  flies  are  exceedingly  numerous  which  act  as 
carriers  of  many  harmful,  and  perhaps  pathogenic  microorganisms. 
Proper  disposal  of  farm  as  well  as  of  city  sewage  implies  a multitude 
of  economic  and  of  engineering  problems,  which  can  be  correctly  solved 
only  if  the  pertinent  bacteriological  facts  are  fully  considered. 

Methods  of  Sewage  Disposal. — Sewage  is  a very  variable  mixture 
of  soluble  and  insoluble  waste  products  of  different  origin  and  of 
unequal  composition.  All  these  substances  are  ultimately  dissolved  and 
transformed  into  mineral  matter  by  bacterial  action,  but  the  time  con- 
sumed for  completing  these  transformations  may  be  short  or  long 
according  to  circumstances.  Highly  diluted  solutions,  such  as  dis- 
charges from  washbasins,  bathtubs,  floor  drains,  etc.,  contain  very 
little  or  no  foul  matter,  and  they  may  be  carried  away  by  run- 
ning water  without  disadvantage.  Human  excrements,  on  the  other 
hand,  as  well  as  other  discharges  rich  in  organic  substances,  as  for 
instance  dairy  wastes,  should  be  kept  separate  and  treated  in  such  a 
manner  as  to  insure  rapid  mineralization  of  the  organic  residues  and 
certain  destruction  of  all  pathogenic  organisms  that  may  be  present. 
Saving  of  the  plant  food  which  these  substances  contain  is  of  consider- 
able importance,  although  frequently  this  point  is  treated  rather 


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lightly.  If  no  complete  sewerage  system  can  be  installed,  the  collection  of 
the  human  excrements  in  properly  constructed  and  well  kept  privies 
deserves  full  consideration.  If  fibrous  peat  or  ashes  are  easily  obtained 
they  may  be  used  for  converting  the  fecal  substances  into  a dry  earth- 
like manure,  which  however  should  he  kept  awray  from  vegetables  and 
berries,  because  of  possible  contamination.  Peat  closets  are  widely  used 
in  Europe ; if  cases  of  typhoid  fever  or  dysentery  occur,  thorough 
disinfection  can  be  accomplished  without  great  difficulty.1  However, 
unless  the  condition  of  a privy  receives  constant  personal  attention 
and  care,  it  is  always  liable  to  become  a nuisance  and  a source  of 
danger  to  the  health  of  man  and  animal. 

The  liquid  wastes  from  a pressure  water  system  can  not  always 
be  carried  away  by  running  water,  and  their  disposal  in  an  ordinary 
seeping  cesspool,  though  still  widely  practiced,  is  strongly  to  be  con- 
demned. If  excretal  matter  is  flushed  away  and  mixed  with  other 
liquid  wastes,  such  sewage  should  always  be  prevented  from  entering 
the  soil  before  it  has  been  properly  treated  to  make  it  as  inoffensive 
and  harmless  as  possible.  Fresh  sewage  is  much  inclined  to  undergo 
anaerobic  decomposition,  and  the  best  place  for  this  is  in  a properly 
constructed  watertight  septic  tank.  After  the  destruction  of  organic 
matter  has  proceeded  therein  to  a point  where  the  cooperation  of 
aerobic  bacteria  is  needed  to  complete  the  mineralization,  the  sewage 
may  be  used  for  irrigation,  or  it  may  be  oxidized  upon  so-called 
trickling  filters,  or  it  may  be  discharged  into  a river. 

The  loss  of  plant  food  makes  the  last-named  arrangement  a practice 
which  is  permissible  only  so  long  as  the  land  itself  is  productive  and 
the  population  not  very  dense.  Eventually  the  conservation  of  all 
plant  food  becomes  more  and  more  imperative ; but  since  the  simple 
and  careful,  though  unhygienic,  handling  of  all  waste  products  as  it 
has  been  practiced  in  China  through  many  centuries,  would  not  find 
favor  with  any  Western  nation,  other  methods  must  be  found  which 
retain  the  advantages  of  the  modern  sewage  treatment,  but  are  equally 
satisfactory  from  the  viewpoint  of  conservation  of  plant  food.  The 
so-called  activated  sludge  process,  recently  introduced  in  American  and 
British  cities,  offers  good  prospects  in  this  direction.  A carefully 
installed  sewerage  system  is  not  too  expensive  for  the  farm,  although 
the  opposite  opinion  is  frequently  held.2 

1 Arbeiten  der  Deutschen  Landivirtschafts-Gesellschaft,  Heft  1;  A.  Stctzer  und 
E.  Herfeldt,  Centralbl.  f . Bakt.,  II.  Abt.,  vol.  1,  1895,  p.  841. 

2 For  detailed  and  illustrated  descriptions  of  the  various  methods  of  sewage  disposal 
see  U.  S.  Public  Health  Service  Bull.  101,  1919,  and  U.  S.  Dept.  Agr.  Farmers’  Bull. 
1227,  1922. 


SEWAGE  DISPOSAL 


217 


Septic  Tanks. — All  sewage  should  be  removed  from  the  farm  build- 
ings through  a water-tight  sewer  to  a water-tight  tank,  where  it  may 
undergo  sedimentation  and  fermentation.  Contamination  of  the  water 
supply  may  take  place  if  foul  matter  is  allowed  to  drain  off  into  the 
ground  from  leaking  pipes  or  from  an  open  cesspool.  Shallow  wells 
are  not  safe  unless  they  are  located  more  than  200  to  500  yards  from 
privies,  stables,  or  other  sources  of  pollution.  Only  where  the  water  has 
to  pass  through  a vertical  column  of  soil  of  more  than  20  feet  depth, 
is  the  complete  removal  of  the  bacteria  to  be  expected.  Since  the  move- 
ment of  the  bacteria-laden  liquids  is  mostly  down-hill,  following  the 
slope  of  the  land,  it  is  very  desirable  to  have  the  well  always  at  a 
higher  level. 

Within  the  septic  tank  all  heavy  insoluble  substances  settle  down 


Fig.  51. — Septic  tank,  after  Kolkwitz. 


as  sludge,  while  the  lighter  particles,  including  all  greasy  material, 
accumulate  on  the  surface  as  a scum,  partly  carried  by  the  gases  which 
are  liberated  in  the  fermentative  processes.  Because  of  the  anaerobic 
conditions  methane  is  produced  in  the  largest  quantities;  the  rest  of 
the  gaseous  mixture  is  made  up  of  hydrogen,  nitrogen,  and  carbon 
dioxide.  Figure  51  shows  a septic  tank  of  simple  construction  with 
its  sludge  deposit  at  the  bottom  and  the  greasy  scum  at  the  surface, 
which  is  prevented  from  escaping  with  the  effluent  by  boards  attached 
to  the  cover.  Naturally,  the  activities  of  the  anaerobic  bacteria  in 
breaking  down  and  dissolving  proteins  and  other  organic  substances 
will  always  give  rise  to  such  offensive  odors  that  it  is  far  better  to 
have  the  septic  tank  not  only  sufficiently  far  away,  but  also  tightly 
covered.  Its  effluent  which  still  contains  large  quantities  of  partly 
dissolved  organic  matter,  should  be  carried  away  underground  to  a 
place  where  final  oxidation  will  be  effected.  Since  the  activities  of 


218 


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the  bacteria  are  disturbed  in  the  ordinary  tank  by  the  inflow  of  fresh 
sewage,  it  is  preferable  to  have  two  chambers,  the  first  one  serving  as 
a settling  chamber  and  the  second  one  as  a fermentation  chamber, 
affording  most  suitable  conditions  for  vigorous  development  and 
activity  of  the  anaerobic  bacteria.  Grease  traps,  sludge  drains,  and 
siphons  for  removing  the  effluent  are  important  practical  improve- 
ments.1 

Irrigation. — If  the  effluent  of  the  septic  tank  is  used  for  irrigation 
the  aerobic  organisms  of  the  soil  will  quickly  destroy  all  organic 
residues  and  mineralize  the  plant  food  contained  therein.  Surface 


Fig.  52. — Trickling  filter,  after  Kolkwitz. 


irrigation,  as  frequently  practiced  in  Europe,  has  great  disadvantages 
especially  during  winter;  subirrigation  or  underground  irrigation  is 
far  superior.  For  this  purpose  the  water  from  the  septic  tank  is 
distributed  by  a system  of  drains  placed  about  one  foot  below  the 
surface  in  porous  ground  to  attain  proper  aeration  and  also  sufficient 
protection  against  frost,  which  can  be  increased,  if  necessary,  by  covering 
the  run  in  cold  weather  with  straw,  hay,  or  leaves.  Permanent  grass 
land  is  generally  best  suited  for  subirrigation.  Deep  underdrainage 
is  necessary,  and  any  well  or  spring  used  for  water  supply  should  be 
located  not  less  than  100  yards  uphill. 

1 For  details  of  constructing  and  operating  septic  tanks  see  U.  S.  Dept,  of  Agr. 
Farmers'  Bull.  1227. 


SEWAGE  DISPOSAL 


219 


Trickling  Filters. — Complete  mineralization  of  all  organic  sub- 
stances by  aerobic  bacteria  can  also  be  attained  by  passing  the  water 
through  a porous  filter  similar  to  that  shown  in  Fig.  52.  It  may  be 
constructed  of  coke,  gravel,  broken  stone,  or  of  other  material,  and 
the  sewage  may  be  applied  either  continuously  by  means  of  a sprinkler, 
or  intermittently  in  so-called  contact  beds.  The  sprinkler  system  offers 
maximum  aeration,  but  good  results  may  be  obtained  also  with  the 
intermittent  system.  While  the  filter  is  filled  for  a few  hours  absorptive 
processes  take  place  which  are  followed  by  vigorous  oxidation,  as  soon 
as  the  liquid  is  drained  off.  Comparative  tests  made  with  dairy  wastes 
showed,  for  instance,  the  following  oxidation  of  organic  substances  in 
per  cent  of  what  had  been  added  d 


Organic 

Matter 

Organic 

Nitrogen 

Fat 

Milk  Sugar 

Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

Sprinkler  system 

73-87 

66-86 

66-98 

100 

Contact  bed 

47-62 

45-62 

87-95 

85-100 

However,  good  results  are  assured  only  if  the  filters  are  carefully 
handled  and  controlled.  By  testing  sewage  and  effluent  with  potassium 
permanganate  the  degree  of  oxidation  can  be  easily  ascertained.  In 
the  experiments  just  mentioned  it  was  93  to  98  in  the  first,  and  58  to 
93  per  cent  in  the  second  ease.  Under  ordinary  farm  conditions  sub- 
irrigation is  undoubtedly  to  be  preferred;  while  for  the  disposal  of 
dairy  wastes  artificial  filters  may  prove  very  helpful.  They  must  be 
covered  to  prevent  odors  and  exclude  flies,  and  in  cold  weather  they 
must  be  warmed  to  15  to  30°  C.,  so  that  the  bacterial  action  can  proceed 
without  interruption. 

Activated  Sludge. — The  so-called  activated  sludge  process  seems  to 
offer  good  prospects  to  the  cities  as  an  efficient  means  of  sewage  dis- 
posal, and  to  the  farmer  as  a valuable  method  of  regaining  the  plant 
food  that  is  brought  daily  from  the  farm  to  the  city  in  form  of  human 
food.  The  activated  sludge  produced  contains  about  5 per  cent 
nitrogen,  of  which  1 / 2 to  2/s  was  found  to  be  easily  available  when  tested 
in  fertilizer  experiments.2 

The  underlying  principle  of  the  activated  sludge  process  consists 

1 A.  Kattein  und  F.  Schoofs,  Milchzeitg.,  vol.  32,  1903,  pp.  98,  114. 

2 Report  of  the  Rothamsted  Experiment  Station,  1918-20,  p.  56;  Third  Ann.  Rep. 
Sewerage  Comm.,  Milwaukee,  Wis. 


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in  replacing  the  anaerobic  fermentations  in  the  ordinary  tank  by  aerobic 
transformations.  For  this  purpose  the  sewage  is  supplied  with  large 
quantities  of  aerobic  bacteria  (in  activated  sludge)  and  with  com- 
pressed air  to  insure  vigorous  action  of  these  organisms.  The  sewage 
circulates  for  several  hours  in  long  or  circular  tanks,  and  the  brown 
jelly-like  mass  which  settles  down,  is  dehydrated  and  ground.  No 
offensive  odors  are  produced  and  the  effluent  is  nearly  potable.  The 
costs  are  high,  but  they  are  partly  covered  by  the  revenues  from  the 
fertilizer  sale.  Not  much  space  is  needed,  and  in  this  as  in  other 
respects  the  activated  sludge  process  recommends  itself  as  an  almost 
ideal  method  of  city  sewage  disposal. 

Chemical  Treatment. — No  complete  purification  of  sewage  is  pos- 
sible by  chemical  means,  but  such  substances  may  be  used  advan- 
tageously for  accelerating  the  settling  of  the  sludge  in  the  tank  and 
for  sterilizing  the  effluent  before  it  is  discharged  into  a stream.  Milk 
of  lime,  alum,  iron  sulfate,  and  sodium  silicate  may  serve  as  clarifiers 
in  settling  tanks ; chlorination  or  ozonization  of  the  effluents  are  the 
most  effective  means  of  eliminating  any  possible  danger  that  might 
be  expected  from  the  presence  of  pathogenic  organisms.  If  fecal  masses 
are  to  be  sterilized  by  chemicals,  as  in  cases  of  typhoid  fever,  very 
large  quantities  are  needed,  otherwise  the  disinfection  will  remain 
incomplete. 


CHAPTER  XIII 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  BARNYARD 

MANURES 

The  gradual  reduction  in  the  natural  fertility  of  all  agricultural 
soils  necessitates  the  application  of  farm  manures  and  of  artificial 
fertilizers.  The  latter  have  been  known  for  little  more  than  one  hundred 
years,  while  barnyard  and  green  manures  have  been  used  for  thousands 
of  years  in  Europe  as  well  as  in  Asia.  At  present  the  application  of 
artificial  fertilizers  is  of  very  great  importance  for  European  agricul- 
ture, because  the  very  dense  population  requires  so  much  food  that 
the  productivity  of  the  soils  must  be  increased  to  the  utmost.  How- 
ever, the  use  of  farm  manures  has  not  lost  its  fundamental  importance, 
and  if  all  the  plant  food  contained  in  human,  animal,  and  plant  residues 
is  carefully  collected  and  given  back  to  the  soil,  enough  food  can  be 
produced  without  the  use  of  artificial  fertilizers  even  in  such  densely 
populated  countries  as  in  China.  There  is  no  doubt  that  in  America 
the  natural  fertility  of  the  soils  could  be  retained  or  restored  to  a 
large  extent  solely  by  the  use  of  farm  manures ; but  widespread  care- 
lessness in  the  handling  of  manure  causes  enormous  losses  in  plant  food 
which  amount  to  several  hundred  million  dollars  annually.  It  is  true 
that  the  high  cost  of  labor  frequently  militates  against  the  extensive 
use  of  bulky  farm  manures,  and  in  such  cases  the  application  of 
artificial  fertilizers  is  no  doubt  preferable.  On  the  other  hand,  it  is 
an  indisputable  fact  that  great  losses  in  humus  content  are  the  main 
cause  of  the  decreased  productivity  of  American  soils,  and  humus  can 
be  restored  only  by  the  consistent  use  of  farm  manures,  while  artificial 
fertilizers  exert  hardly  any  beneficial  effect  in  this  respect.  Animal 
manures  are  of  special  importance,  because  they  not  only  contain 
large  quantities  of  organic  substances,  but  also  large  numbers  of 
bacteria  and  related  microorganisms,  which  participate  in  the  humus 
formation  and  in  other  transformations  of  plant  food  in  the  soil.1 

1 For  references  see  F.  Lohnis,  “Handbuch  der  landw.  Bakteriologie,”  Chap.  IV. 


221 


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TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


1.  GERM  CONTENT  OF  BARNYARD  MANURES 

The  solid  excrements  of  animals  are  made  up  of  partly  decomposed 
food  residues  and  of  the  bacteria  that  cause  their  decomposition. 
Unstained  smears  give  very  interesting  pictures  like  that  shown  in 
Fig.  53.  The  weight  of  living  and  of  dead  bacteria 
in  the  feces  was  found  to  be  equal  to  1/10  to  1/5 
of  the  weight  of  the  dung ; calculated  on  the  basis 
of  fresh  weight  the  number  of  living  cells  would 
approximate  20,000  to  40,000  millions  per  g.  If 
plate  cultures  are  made,  usually  no  more  than  a 
few  hundred  millions  per  g.  will  grow,  but  such 
results  are  of  restricted  value,  because  many  of 
the  fecal  bacteria  refuse  to  grow  on  the  plates. 
From  human  feces  as  many  as  18,000  millions  per 
g.  have  been  cultivated.  Fresh  urine  contains,  as 
a rule,  comparatively  few  microorganisms,  and  those  present  in  the 
litter  are  also  of  minor  importance,  so  far  as  numbers  are  concerned; 
hut  in  regard  to  the  activities  performed  by  the  different  groups  of 
microorganisms  the  bacteria  growing  in  urine  as  well  as  those  on  the 
straw  are  as  essential  as  those  in  the  feces. 

Frequency  of  Microorganisms  in  Manure. — The  total  germ  content 
of  barnyard  manure  varies  widely  according  to  the  composition  and 
the  age  of  the  material.  Many  bacteria  are  already  present  in  fresh 
stable  manure,  but  if  this  is  kept  for  several  weeks  or  months,  at  first 
a marked  multiplication  takes  place  which  is  followed  by  a gradual 
decline.  Plate  counts  gave,  for  instance,  the  following  results  whose 
relations  are  of  greater  interest  than  the  numbers  themselves  because 
of  the  reason  discussed  above.  From  fresh  material  were  grown  in 


millions  per  g.  d 

Feces 

Urine 

Straw 

390-480 

1-2 

1,  3-19 

These  materials  were  mixed  as  in  average  manure  in  a relation  of  7 
parts  feces  to  l1/4  part  urine  to  1 part  straw,  and  kept  for  six  weeks 
at  20°  C.  Then  the  plate  counts  showed  in  millions  per  g. : 

In  Feces  and  Straw  In  Feces,  Urine,  and  Straw  In  Urine  Alone 

4800-5700  11,000-11,600  3 

As  was  to  be  expected,  the  bacterial  growth  was  much  more  vigorous 
in  the  mixture  made  up  of  feces,  urine,  and  straw,  than  in  feces  and 
1 F.  Lohnis  and  J.  H.  Smith,  Fuhling’s  landw.  Zeitg.,  vol.  63, 1914,  p.  153. 


Fig.  53. — Smear  made 
from  cow  dung,  un- 
stained (X700). 


MICROORGANISMS  IN  BARNYARD  MANURES 


223 


straw  without  urine,  or  in  urine  alone.  Nevertheless,  feces  and  straw 
alone  showed  also  a marked  increase  in  numbers,  which  fact  is  of 
special  importance  because  certain  reasons  make  it  desirable  to  keep 
solid  and  liquid  manures  separate  as  much  as  possible. 

It  may  be  assumed  that  100  lbs.  of  ordinary  barnyard  manure  con- 
tain approximately  1 to  iy2  lbs.  of  living  bacteria  and  fungi.  If  15 
tons  of  manure  are  applied  to  one  acre  of  land,  no  less  than  300  to  450 
lbs.  of  living  matter  are  incorporated  in  the  soil  together  with  about 
6000  lbs.  of  organic  matter.  It  is  easily  understood  that  the  biological 
conditions  in  the  soil  are  greatly  affected  by  such  a treatment. 

Groups  of  Microorganisms  in  Manure. — The  very  complex  mixture 
of  all  kinds  of  organic  residues  stimulates  the  development  of  prac- 
tically all  groups  of  microorganisms.  The  anaerobic  B.  putrificus  and 
related  forms  work  together  with  B.  fluorescens,  proteus,  and  other 
aerobic  species  in  dissolving  and  disintegrating  proteins  and  other 
nitrogenous  compounds.  B.  Pasteuri  and  many  other  rod-shaped  as 
well  as  coccoid  bacteria  transform  the  urine  nitrogen  to  ammonia. 
Anaerobic  butyric  acid  bacilli,  aerobic  sporulating  bacilli,  B.  coli,  B. 
aerogenes,  and  various  streptococci  are  breaking  down  the  remaining 
soluble  carbohydrates,  while  pectic  substances  and  cellulose  are 
attacked  by  special  groups  of  organisms  which  are  again  partly 
anaerobic  and  partly  aerobic.  Denitrifying  bacteria  are  frequent  in  all 
manures,  whereas  nitrifying  organisms  are  absent  in  fresh  and  not 
very  numerous  in  old  manure.  Anaerobic  and  aerobic  nitrogen  fixing 
bacteria  are  equally  present.  The  Actinomycetes  constitute  another 
characteristic  group  of  manure  organisms,  and  the  same  is  true  of  yeasts 
and  molds.  In  dry  manure  heavy  mold  growth  is  always  visible  with  the 
naked  eye.  Higher  fungi,  too,  grow  well  on  rotted  manure,  as  is  demon- 
strated by  artificial  mushroom  cultures.  Protozoa  are  frequent,  and  some 
other  interesting  groups,  the  Myxomycetes  and  the  Myxobacteria,  also 
grow  well  on  manure.  The  latter  are  characterized  by  a curious  mode 
of  colony  formation  and  of  fructification,1  which  make  them  of  great 
systematic  and  physiological  interest ; but  they  are  of  minor  importance 
as  far  as  the  biochemical  transformations  in  the  manure  are  concerned. 

If  pathogenic  organisms  get  into  the  manure,  as  may  easily  happen 
in  cases  of  foot  and  mouth  disease,  mastitis,  and  tuberculosis,  they 
may  remain  alive  and  may  even  multiply  to  some  extent,  and  such 
manure  is  very  liable  to  become  a source  of  new  infection.  It  is  possible 
to  destroy  these  pathogenic  organisms  in  the  manure  by  piling  it  in 

1 R.  Thaxter,  Boian.  Gazette,  vol.  17,  1892,  p.  389;  vol.  23,  1897,  p.  395;  vol.  37, 
1904,  p.  405.  Striking  colored  pictures  were  published  by  Quehl  in  Centralbl.  f.  Baki., 
II.  Abt.,  vol.  16,  1906,  pp.  9-34. 


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loose  high  heaps  wherein  the  temperature  quickly  rises  to  about  70° 
C.,1  but  under  ordinary  farm  conditions  such  a treatment  would  be 
of  doubtful  value ; if  the  highly  resistant  spores  of  the  anthrax  bacillus 
are  present,  it  would  be  quite  insufficient.  Thorough  disinfection  with 
strong  acids,  or  the  destruction  of  infected  manure  by  fire  are  the  only 
reliable  means  of  rendering  such  material  harmless. 


2.  BACTERIAL  ACTIVITIES  IN  BARNYARD  MANURES 

It  is  a well  known  fact  that  the  chemical  composition  of  animal 
manures  shows  wide  variations,  and  still  more  irregular  are  the  effects 
realized  from  the  application  of  barnyard  manures.  In  field  experi- 
ments of  four  years  duration  the  following  quantities  of  nitrogen, 
phosphorus,  and  of  potassium  were  recovered  in  the  manured  crops:2 

Nitrogen  Phosphorus  Potassium 

Per  Cent  Per  Cent  Per  Cent 

7.8—46.1  10.1—75.6  22.4—85.1 

Similar  results  have  been  obtained  quite  generally,  but,  as  a rule,  no 
investigations  have  been  made  to  find  out  why  the  effects  did  show 
such  wide  variations.  Since  the  manure  produced  annually  represents 
an  economic  value  of  approximately  $50  per  full  grown  animal,  it  is 
rather  disappointing  that  so  little  has  been  done  thus  far  to  discover 
the  chemical  and  biological  reasons  for  these  wide  differences.  It  is 
beyond  doubt  that  after  such  investigations  will  have  been  made,  and 
the  conditions  are  known  under  which  a high  fertilizing  effect  is 
assured,  the  intelligent  application  of  barnyard  manure  will  prove 
much  more  profitable  than  is  frequently  the  case  to-day. 

The  Rotting  of  Manure. — There  is  a rather  widespread  belief  that 
it  is  best  to  apply  the  manure  as  soon  as  it  is  made ; losses  which  may 
occur  during  storage  are  thus  excluded,  and  all  plant  food  is  given 
back  to  the  soil.  However,  this  practice  gives  satisfactory  results 
only  if  sufficient  time,  at  least  6 to  8 weeks,  will  elapse  before  a new 
crop  is  planted  on  the  manured  field.  Otherwise  the  undecomposed 
organic  substances  will  prove  distinctly  harmful  to  the  crop,  because 
they  stimulate  the  bacterial  assimilation  of  nitrate,  ammonia,  and 
amino  nitrogen  in  the  soil,  and  thereby  cause  a nitrogen  starvation 
of  the  higher  plants.  For  example,  the  following  percentage  of  manure 
nitrogen  was  taken  up  by  four  successive  crops,  -when  the  same  mix- 
tures that  were  used  for  making  the  bacterial  counts  mentioned  above, 

1 H.  Bohtz,  Arbeiten  a.  d.  Kais.  Gesundh.  Amte,  vol.  33,  1910,  p.  313. 

2 Arbeiten  d.  Deutschen  Landw.-Gesellschaft,  Heft  198,  1911. 


MICROORGANISMS  IN  BARNYARD  MANURES 


225 


were  applied  fresh  or  after  they  had  undergone  a rotting  of  six  weeks 
duration  im- 


Per Cent  N Recovered 

Feces  and  Straw 

Feces,  Urine, 
and  Straw 

Urine  Alone 

Fresh  manure 

- 3.2 

+ 14.7 

+56.7 

Six  weeks  old 

+ 17.3 

+40.1 

+73.5 

partial  decomposition  of  the  organic  substances  proved  highly  benefi- 
cial in  every  case,  and  special  tests  confirmed  that  the  crops  were  much 
better  supplied  with  nitrate  if  manure  six  weeks  old  was  used. 

Great  numbers  of  analogous  observations  have  been  recorded  in 
the  literature.  Two  months  rotting  permits  the  removal  of  harmful 
carbonaceous  substances  and  the  initial  decomposition  of  part  of  the 
nitrogenous  compounds ; immediately  after  such  manure  has  been 
plowed  under  nitrification  sets  in.  Nevertheless,  the  labor  problem 
and  the  certainty  that  large  losses  in  plant  food  will  occur  if  the 
manure  is  not  stored  and  handled  very  carefully,  may  make  it  advisable 
to  apply  the  manure  as  soon  as  possible  after  it  is  made.  If  the  new 
crop  is  not  planted  before  2 or  3 months  have  elapsed,  and  if  the 
temperature  is  not  too  low,  the  necessary  transformations  will  take 
place  in  the  soil  itself.  Some  weeks  of  “ripening”  before  application 
also  increases  the  beneficial  effect  of  liquid  manure;  if  used  fresh, 
“burning”  of  the  plants  is  often  noticeable. 

Transformation  of  Carbohydrates. — A loss  of  10  to  30  per  cent  of 
the  total  solids  is  always  to  be  expected  during  the  rotting  of  manure. 
It  is  mainly  caused  by  the  fermentation  of  carbohydrates.  If  manure 
is  carelessly  stored  or  kept  for  a very  long  time,  the  losses  may  reach 
50  to  70  per  cent  of  the  total  dry  weight.  The  percentage  of  soluble 
carbohydrates  is  lowest  as  far  as  the  composition  of  fresh  manure  is 
concerned,  but  in  regard  to  the  rate  of  decomposition  they  rank  first. 
Next  come  the  pectic  substances,  while  the  cellulose  is  relatively  most 
resistant  and  present  in  largest  quantities.  Comparative  tests  have 
shown  the  following  relations  in  the  extent  to  which  these  three  groups 
of  carbohydrates  are  being  broken  down  during  the  normal  rotting 
of  manure.  Of  the  total  quantities  originally  present  the  following 
percentages  underwent  fermentation: 

Soluble  Carbohydrates  Pectic  Substances  Cellulose 

20  to  30  per  cent  15  to  20  per  cent  7 to  10  per  cent 

1 F.  Lohnis  and  J.  H.  Smith,  1.  c. 


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Because  all  these  substances  may  be  attacked  by  aerobic  as  well  as 
by  anaerobic  bacteria,  it  does  not  make  much  difference  whether  the 
fermentations  take  place  in  the  presence  or  in  the  absence  of  air. 
Under  otherwise  equal  conditions  the  quantities  transformed  are  ap- 
proximately the  same.  But  very  great  differences  become  apparent 
if  samples  of  the  same  manure  are  allowed  to  rot  at  low  or  at  high 
temperatures.  Usually  the  losses  in  carbohydrates  are  4-  to  8-fold 
larger  at  35°  than  at  15°  C.  Since  heat  is  generated  in  the  fermen- 
tative processes  themselves,  it  is  of  great  importance  to  keep  the 
manure  sufficiently  moist  and  cool. 

All  kinds  of  organic  acids  are  produced  in  the  decomposition  of 


Fig.  54. — Cellulose  agar  culture  (§  nat.  size),  (a)  before,  ( b ) after  treatment  with 

hydrochloric  acid. 


carbohydrates.  Butyric  acid  plays  an  important  role  among  them; 
it  is  partly  responsible  for  the  peculiar  odor  of  rotted  manure.  If  the 
am  mollification  remains  low,  the  acid  formation  may  be  followed  by 
the  development  of  a distinctly  acid  reaction  in  the  manure. 

Frequency  and  Activity  of  Cellulose  Bacteria. — There  is  little  known 
at  present  about  the  actual  numbers  of  aerobic  and  anaerobic  cellulose 
destroying  bacteria  in  manure.  That  they,  too,  multiply  rapidly  dur- 
ing the  first  few  weeks  has  been  ascertained  by  J.  H.  Smith.1  who 
observed  a ten-fold  increase  within  six  weeks.  But  more  important 


1 F.  Lohnis  and  J.  H.  Smith,  1.  c. 


MICROORGANISMS  IN  BARNYARD  MANURES 


227 


than  this  increase  in  numbers  of  microorganisms  is  the  fact  that  the 
active  enzymes  are  soluble  and  exert  a far-reaching  effect  even  where 
the  bacteria  do  not  grow,  or  after  they  have  died.  In  Fig.  54  a cellulose 
agar  plate  culture  is  shown  in  which  very  minute,  nearly  invisible 
colonies  of  aerobic  cellulose  bacteria  are  surrounded  by  comparatively 
large  clear  zones.  Their  appearance  is  similar  to  that  produced  by 
the  growth  of  lactic  acid  bacteria  in  sugar  agar  containing  chalk  (Fig. 
44,  p.  177).  However,  it  is  not  the  dissolution  of  added  chalk,  but  the 
disintegration  of  the  cellulose  present  in  the  agar  that  causes  the  forma- 
tion of  these  clear  zones.  When  the  plate  was  treated  with  hydrochloric 
acid  all  chalk  Avas  dissolved,  but  the  clear  areas  were  left  unchanged.1 

Gas  Formation. — The  gases  liberated  in  the  transformation  of 
carbohydrates  and  of  other  organic  substances  are  usually  composed 
of  approximately  equal  parts  of  carbon  dioxide  and  methane.  In 
general  not  much  hydrogen  is  found,  although  it  is  produced  in  con- 
siderable quantities  in  the  decomposition  of  carbohydrates  by  the  fecal 
lactic  acid  bacteria  (B.  coli  and  B.  aerogenes)  and  by  the  anaerobic 
butyric  acid  bacilli  (B.  amylobacter).  The  reason  why  it  is  so  rare 
in  the  gases  escaping  from  the  manure  is  that  in  secondary  reactions, 
taking  place  in  the  anaerobic  transformations  of  carbohydrates  and 
of  fatty  acids,  methane  producing  bacteria  are  making  use  of  the 
hydrogen.  Even  carbonates  can  serve  as  a source  of  methane  in  the 
presence  of  hydrogen.2 

Since  usually  20  per  cent  or  more  of  the  organic  substances  are 
destroyed  in  the  rotting  manure,  it  can  be  easily  calculated  that  very 
large  quantities  of  gases  are  produced.  If  it  is  assumed  that  1 cubic 
meter  of  manure  contains  250  kg.  of  water-free  organic  substances, 
and  20  per  cent  of  them,  that  is,  50  kg.  containing  25  kg.  carbon,  are 
destroyed,  approximately  46  kg.  carbon  dioxide  and  16.7  kg.  methane 
would  be  produced,  equal  to  2 X 23.5  = 47  cubic  meters  of  gas.  In 
actual  tests  made  the  quantities  measured  were  between  10  and  100 
cubic  meters  of  gases  per  1 cubic  meter  of  manure.  Accordingly,  all 
air  spaces  within  a large  manure  pile  are  filled  with  carbon  dioxide 
and  methane.  Such  a condition  is  distinctly  favorable  to  the  activities 
of  anaerobic  organisms,  and  at  the  same  time  the  e\mporation  of 
ammonia  is  materially  reduced,  because  ammonium  carbonate  is  much 
more  stable  in  an  atmosphere  of  carbon  dioxide  than  in  the  presence 
of  air. 

1 Microscopic  pictures  of  such  plates  were  published  in  a paper  by  F.  Lohnis  and 
Gr.  Lochhead,  Centralbl.f.  Bakt.,  II.  Abt.,  vol.  37,  1913,  p.  490. 

2 N.  L.  Sohngen,  Recueil  des  Travaux  chimiques  des  Pays-Bas,  etc.,  2.  ser.,  vol.  14, 
1910,  p.  238. 


228 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


Humus  Formation. — The  color  of  old  manure  shows  that  part  of 
the  organic  compounds  is  transformed  into  brown  or  black  soluble  and 
insoluble  substances,  commonly  called  humus.  Although  much  remains 
to  be  investigated,  it  is  certain  that  only  a restricted  humification  is 
desirable  in  manure.  It  is  known  from  the  great  resistance  of  peat 
that  humus  produced  under  anaerobic  conditions  is  of  little  value  as 
a source  of  plant  food,  and  the  same  is  true  of  the  black  tough  manure 
as  it  occurs  in  the  bottom  layers  of  big  manure  piles  that  have  been 
kept  for  a long  time.  Such  material  is  often  plowed  up,  practically 
unchanged,  after  a full  season  in  the  soil.  Ultimately  the  plant  food 
inclosed  in  such  peaty  flakes  will  become  available,  but  a prompt 
fertilizing  effect  can  not  be  expected.  Undoubtedly,  the  interaction 
between  amino  acids  and  carbohydrates,  which  was  discussed  on  p.  128, 
plays  a considerable  role  in  the  humus  formation  in  manure  as  it  does 
in  soil.  Free  ammonia,  produced  in  the  mineralization  of  organic 
nitrogenous  compounds,  causes  also  a brown  discoloration  of  the  straw. 

Production  of  Heat. — The  rapid  decomposition  of  carbonaceous  com- 
pounds invariably  causes  a rise  in  temperature  in  the  manure  pile. 
If  the  material  is  loose  and  relatively  dry,  as  in  horse  manure,  mostly 
aerobic  processes  take  place,  and  the  production  of  heat  is  very  pro- 
nounced. In  hot  frames  practical  application  is  made  of  this  process. 
Under  anaerobic  conditions,  as  a rule,  much  less  heat  is  generated. 
Complete  oxidation  of  glucose  to  carbon  dioxide  and  water  liberates, 
for  instance,  sixteen  times  as  many  calories,  as  are  produced  in  the 
anaerobic  transformation  to  carbon  dioxide  and  methane.  However, 
barnyard  manure  is  such  a complex  mixture  that  a direct  application 
of  simple  physico-chemical  relations  can  not  be  made.  In  fact, 
it  has  been  repeatedly  recorded  that  despite  a stronger  heat  production 
the  loss  in  organic  substances  was  actually  smaller  than  in  another 
instance  where  the  increase  in  temperature  was  less  conspicuous.  Rela- 
tively dry  manure  loosely  stacked  reaches  almost  regularly  65  to  70° 
C.,  while  tightly  packed  material,  containing  approximately  80  per  cent 
moisture,  does  not  easily  go  above  40°  C.  Excessive  heat  production 
indicates  large  losses  in  valuable  material  in  the  manure  pile  as  in 
the  ensiled  fodder ; it  may  lead  to  the  formation  of  coal-like  substances 
that  are  of  very  little  value.  Thermophilic  and  thermogenic  organisms 
are  frequent  in  every  manure,  but  they  can  not  do  much  harm  if  the 
manure  is  kept  tight  and  well  moistened. 

Transformation  of  Nitrogenous  Compounds. — The  majority  of 
analyses  made  of  manures  show  nothing  but  the  amount  of  total 
nitrogen  and  perhaps  its  gradual  changes.  In  view  of  the  different 
origin  and  the  complex  nature  of  the  constituents  of  barnyard  manure, 


MICROORGANISMS  IN  BARNYARD  MANURES 


229 


very  little  insight  can  be  gained  from  such  analyses.  In  fact,  not 
much  is  known  concerning  the  nitrogen  metabolism  of  animal  manure, 
despite  its  very  great  economic  value.  The  nitrogen  present  in  solid 
excrements  and  in  the  litter  (straw,  peat,  leaves,  etc.)  is  incorporated 
mostly  in  complex,  insoluble,  protein-like  compounds  which  offer  great 
resistance  to  the  attacks  of  bacteria  and  are,  therefore,  very  slowly 
mineralized.  In  urine,  on  the  other  hand,  practically  all  of  the  nitrogen 
is  present  in  soluble  form,  as  urea,  hippuric  acid,  and  to  a small  extent 
as  uric  and  glycin-phenylacetie  acids.  All  these  amino  compounds  are 
more  or  less  easily  transformed  to  ammonia ; but  in  the  presence  of 
large  quantities  of  soluble  carbonaceous  substances  the  opposite  process 
may  predominate,  that  is,  ammonia  and  amino  nitrogen  may  be 
assimilated  by  microorganisms  and  transformed  to  resistant  protein 
substances.  To  what  extent  the  addition  of  urine  may  influence  the 
nitrogen  metabolism  in  manures  may  be  seen  from  the  following  data, 
recorded  by  E.  B.  Yorhees  and  J.  G.  Lipman1  with  fresh  and  rotted 
manures : 


Percentage  of  Total  Nitrogen 

Organic  Nitrogen 

Ammonia 

Insoluble 

Soluble 

Nitrogen 

f fresh 

Feces  and  straw  only  j ro^e(j 

85.0—86.2 

76.8—83.2 

6.1—  7.4 
7.9—10.1 

7.6—  7.7 
8.9—13.1 

f fresh 

Feces,  straw,  and  urine  ^ ^ 

45.6—51.6 

62.4—67.1 

15.8—17.2 
8.2—  9.5 

32 . 6—37 . 7 
23.4—29.4 

The  solubility  of  the  organic  nitrogen  showed  an  increase  in  the  first, 
and  a decrease  in  the  second  case;  the  same  holds  true  in  regard  to 
ammonia  nitrogen. 

If  samples  of  the  same  material  are  kept  under  aerobic  and  under 
anaerobic  conditions,  usually  the  insoluble  organic  nitrogen  increases 
in  the  aerobic  samples,  and  decreases  in  the  anaerobic  samples;  the 
ammonia  content  rises  more  rapidly  in  the  absence  of  air,  and  the 
losses  in  total  nitrogen  are  much  larger  under  aerobic  conditions.  Con- 
sidering all  these  variable  factors  it  is  easily  understood  why  the 
nitrogen  contents  of  barnyard  manures  exhibit  very  great  differences 
in  quantity  as  well  as  in  quality.  The  following  data  illustrate  this 
point : 

1 Annual  Report  N.  J.  Agr.  Exp.  Stat.,  vol.  26,  1905,  pp.  140,  161. 


230 


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Percentage 

Manures  from 

Horses 

Cattle 

Sheep 

Total  nitrogen 

Amino  nitrogen 

Ammonia  nitrogen 

0.39—0.70 
0 —0.07 

0.07—0.34 

0.34—0.65 
0 —0.08 
0.04—0.20 

0.55—1.22 

0.03—0.14 

0.22-0-47 

Close  relations  between  ammonium  content  and  fertilizing  effect  of 
stable  manures  have  been  observed  frequently,  though  not  regularly. 
This  explains  in  part  why  either  very  satisfactory,  or  rather  unsatis- 
factory results  are  obtained  by  the  application  of  different  farm 
manures,  whose  chemical  composition,  as  a rule,  is  unknown. 

Ammonification.  Feces  and  straw  show  little  ammonification ; but 
this  process  is  very  strong  in  urine.  Comparative  tests  with  such 
material  held  for  six  weeks  at  20°  C.  furnished  the  following  results  d 


Feces  and- Straw 

Feces,  Straw, 
and  Urine 

Urine  Alone 

Per  cent  of  total  nitrogen 

transformed  to  ammonia. . . 

2-2.8 

28.4-30 

75-80 

Even  if  the  tests  were  continued  for  a much  longer  time,  not  more 
than  1/5  of  the  nitrogen  in  mixtures  made  up  of  feces  and  straw  was 
transformed  to  ammonia.  The  following  facts  explain  this  high  re- 
sistance. Dead  and  living  bacteria  constitute  10  to  20  per  cent  of  the 
dry  solids  of  feces,  whose  total  nitrogen  content,  as  a rule,  was  found 
to  be  2 to  3 per  cent,  while  dried  bacteria  contain  usually  about  10  per 
cent  of  nitrogen.  Accordingly,  approximately  one  half  of  the  total 
nitrogen  of  the  solid  excrements  is  incorporated  in  bacterial  cells.  One 
half  of  the  bacteria  is  alive,  hence  this  nitrogen  is  not  available.  The 
other  half  is  dead,  but  the  mineralization  of  bacterial  proteins  pro- 
ceeds rather  slowly,  as  was  pointed  out  before  (p.  106).  The  nitrogen 
left  in  the  indigested  food  residues  is,  of  course,  very  resistant,  other- 
wise the  powerful  digesting  enzymes  of  the  animal  organism  would 
have  made  use  of  it.  Straw  contains  but  little  nitrogen,  and  not  more 
than  y4  of  it  is  digested  by  pepsin  in  the  presence  of  hydrochloric  acid. 

i F.  Lohnis  and  J.  H.  Smith,  1.  e. 


MICROORGANISMS  IN  BARNYARD  MANURES 


231 


Furthermore,  its  relatively  high  carbon  content  is  not  favorable  for 
a vigorous  ammonification. 

The  amino  nitrogen  in  urine,  on  the  other  hand,  is  practically  com- 
pletely transformed  to  ammonia  within  a few  weeks.  If  no  loss  occurs 
by  evaporation,  80  to  90  per  cent  or  more  of  the  total  nitrogen  of  well 
fermented  liquid  manure  is  present  as  ammonium  carbonate,  as  organic 
ammonium  salts,  or  as  free  ammonia.  Because  in  the  transformation 
of  hippuric  acid  relatively  large  quantities  of  benzoic  acid  are  split 
oft;  (see  p.  100),  liquid  manuring  adds  to  every  acre  of  land  several 
hundred  pounds  of  benzoic  acid  and  a smaller  quantity  of  phenols.  It 
is  obvious  that  the  resulting  change  in  the  composition  of  the  soil 
solution  must  exert  a considerable  influence  upon  the  microflora  of 
the  soil. 

Assimilation  of  Amino  and  Ammonia  Nitrogen. — If  urine  is  mixed 
with  feces  and  straw  a profound  change  in  the  nitrogen-carbon  ratio 
takes  place  ; ammonification  is  more  or  less  checked,  and  in  the  presence 
of  air  the  assimilation  of  ammonia  and  amino  nitrogen  is  greatly 
stimulated.  The  analyses  made  by  Vorhees  and  Lipman,  quoted  above, 
demonstrate  these  facts  very  clearly ; ammonia  and  soluble  organic 
nitrogen  decrease  in  the  complete  mixture,  while  the  insoluble  nitrogen 
increases.  Opposite  relations  prevail  in  the  mixture  free  of  urine. 
30  to  70  per  cent  of  the  urine  nitrogen  may  be  assimilated  if  the 
liquid  excreta  are  mixed  with  feces  and  straw.  It  Avas  mentioned 
before  that  many  bacteria  and  fungi  are  enabled  to  assimilate  all 
soluble  ammonia  and  amino  nitrogen  within  a feAv  days.  These  organ- 
isms are  generally  more  active  in  the  presence  of  air,  but  partial 
assimilation  takes  place  also  in  the  absence  of  air. 

If  peat  is  used  instead  of  straAv,  more  of  the  ammonia  is  retained, 
but  some  assimilation  still  occurs,  and  part  of  the  ammonia  escapes 
by  evaporation.  It  is  much  better  to  collect  and  to  use  liquid  manure 
separately  from  the  mixture  of  solid  excrements  and  straAv.  In  liquid 
manure  the  ammonification  proceeds  undisturbed  by  those  retrograde 
transformations,  and  the  best  manuring  effect  is  assured.  WhereA'er 
the  cost  of  labor  and  other  economic  conditions  permit,  this  system 
should  be  adopted. 

Nitrification. — As  a rule,  direct  nitrate  determinations  in  manure 
give  almost  or  completely  negative  results,  but  this  does  not  pro\re 
that  nitrification  is  entirely  absent.  Here  the  situation  is  similar  to 
that  noted  with  ammonia.  As  soon  as  ammonia  is  produced,  it  may 
evaporate  or  may  be  assimilated,  and  nitrite  and  nitrate  may  be  de- 
stroyed by  denitrifying  bacteria  or  may  be  reduced  and  assimilated. 
The  conditions  under  which  vigorous  nitrification  takes  place,  are 


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(1)  the  presence  of  ammonium  salts,  but  the  absence  of  considerable 
quantities  of  free  ammonia,  (2)  full  aeration,  (3)  the  absence  of  large 
quantities  of  soluble  organic  substances,  (4)  the  presence  of  basic  sub- 
stances for  neutralizing  the  acids.  Urine  alone,  or  mixed  with  feces 
and  straw,  tends  to  suppress  the  nitrification  because  of  its  relatively 
large  content  of  soluble  organic  substances  and  of  free  ammonia. 
Feces  and  straw  moistened  with  water  offer  more  suitable  conditions 
to  the  nitrifying  organisms,  which  are,  in  fact,  quite  active  in  the 
surface  layers  of  such  manure  in  the  pile,  or  after  it  has  been  spread 
on  the  field.  If  leaching  is  prevented  a gradual  increase  in  nitrate 
nitrogen  may  be  observed,  and  even  if  the  nitrate  is  washed  away 
the  nitrification  is  indicated  by  the  multiplication  of  nitrifying  organ- 
isms, which  are  always  scarce  in  fresh  manure.1  Solid  and  liquid 
excrements  are  practically  free  from  nitrite  and  nitrate  bacteria,  straw 
or  other  litter  may  contain  few  of  them,  but  a regular  source  of  infec- 
tion is  the  old  dirt  present  on  stable  floors,  in  the  barnyard,  etc.  When 
tested  in  soil  or  in  ammonium  sulfate  solution  such  material  gives 
prompt  niti’ification.2 

Losses  of  Nitrogen. — Nitrogen  escapes  from  the  manure  pile  into 
the  air  either  as  free  ammonia  or  in  elementary  form.  Losses  of  non- 
volatile nitrogenous  compounds  by  leaching  should  always  be  pre- 
vented, of  course.  A certain  amount  of  ammonia  is  lost  while  the 
manure  is  still  in  the  stable,  because  a heavy  infection  of  the  urine 
takes  place  as  soon  as  this  comes  into  contact  with  the  floor,  and  the 
transformation  of  urea  sets  in  almost  immediately.  These  losses  can 
be  kept  down  by  the  use  of  peat ; sawdust  and  straw,  on  the  other  hand, 
stimulate  the  evaporation  because  of  their  relatively  large  surfaces  and 
low  absorptive  powers.  In  comparative  tests,  for  instance,  the  following 
losses  in  per  cent  of  the  total  nitrogen  have  been  observed,  when  the 
manure  was  kept  one  day  in  the  stable  :3 

Peat  Litter  Sawdust  Straw  Litter 

7.1  per  cent  11.1  per  cent  19.8  per  cent 

Naturally,  the  evaporation  of  ammonia  continues  when  the  manure 
is  removed  from  the  stable.  If  it  is  spread  on  the  field,  further  losses 
will  be  more  or  less  completely  prevented  by  the  absorptive  power 
of  the  soil,  but  in  the  manure  pile  the  volatilization  will  continue 

1 B.  Niklewski,  Centralbl.f.  Bakl.,  II.  Abt.,  vol.  26, 1910,  pp.  388-442;  E.  J.  Russell 
and  E.  H.  Richards,  Jour.  Agr.  Science,  vol.  8,  1917,  pp.  495-563. 

2 F.  Lohnis  and  H.  J.  Smith,  1.  c. 

3 Hj.  von  Feilitzen,  Svenska  Mosskulturforen.  Tidskrift,  vol.  24,  1910,  p.  10. 


MICROORGANISMS  IN  BARNYARD  MANURES 


233 


to  reduce  the  ammonia  content  of  the  manure,  especially  if  urine  was 
mixed  with  feces  and  straw. 

In  addition  to  the  escape  of  nitrogen  in  form  of  ammonia  frequently 
large  losses  in  elementary  form  have  been  observed.  Their  causes  are 
not  yet  fully  understood,  but  it  is  certain  that  such  losses  play  a 
conspicuous  role  only  in  the  presence  of  air. 

Liberation  of  Nitrogen. — Denitrification  is  the  only  well-known 
process  by  which  nitrogen  may  be  liberated  from  the  manure  in  ele- 
mentary form.  Since  ammonia  can  not  be  nitrified  in  the  absence  of 
air,  denitrification  can  be  completely  prevented  if  the  air  is  excluded 
by  placing  the  manure  in  tightly  covered  water-tight  pits  as  soon  as 
it  is  removed  from  the  stable.  If  the  manure  pile  is  fully  exposed 
to  the  air,  as  is  usually  done,  nitrification  will  occur  in  the  surface 
layers,  and  the  nitrites  and  nitrates  will  be  decomposed  if  they  are 
either  washed  down  into  deeper  layers  where  air  is  absent,  or  if 
anaerobic  conditions  are  established  by  placing  fresh  layers  of  manure 
on  top.  Denitrifying  bacteria  and  carbohydrates  are  present  in 
abundance  in  every  manure  pile,  and  denitrification  will  always  occur 
if  nitrification  is  not  prevented. 

However,  losses  of  nitrogen  in  elementary  form  have  also  been  ob- 
served under  conditions  where  nitrification  could  not  be  the  indirect 
cause.  The  mechanism  of  these  processes  remains  to  be  investigated; 
probably  unstable  compounds  are  formed  in  the  decomposition  of  the 
organic  nitrogenous  compounds  which  break  down  to  elementary 
nitrogen.1  Whether  or  not  free  ammonia  can  be  directly  oxidized 
by  bacteria  to  water  and  elementary  nitrogen,  as  some  authors  have 
assumed,  is  another  question  that  can  not  be  answered  at  present. 
Altogether,  it  is  much  to  be  regretted  that  so  little  accurate  work 
has  been  and  is  being  done  in  regard  to  this  very  important  problem. 
Year  after  year  enormous  economic  losses  continue  to  occur,  and  very 
little  is  done  to  prevent  them. 

Fixation  of  Nitrogen. — Occasionally  instead  of  a loss,  a gain  in 
nitrogen  has  been  found  when  the  nitrogen  balance  was  determined 
in  samples  of  rotting  manure.2  Several  nitrogen  fixing  bacteria  have 
been  isolated  from  such  material.  Most  common,  of  course,  are  those 
belonging  to  the  group  of  B.  amylobacter;  but  Azotobacter,  Bact.  lactis 
viscosum,  and  other  aerobic  organisms  capable  of  assimilating  ele- 
mentary nitrogen  have  also  been  discovered  in  manure.  Because  of 
their  frequency  in  soil  this  is  by  no  means  surprising,  and  the  large 

1 Russell  and  Richards,  1.  c. 

2 W.  E.  Tottingham,  Jour.  Biol.  Chem.,  vol.  24,  1916,  p.  221;  E.  H.  Richards, 
Jour.  Agr.  Science,  vol.  8, 1917,  p.  299. 


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quantities  of  carbonaceous  material  always  present  in  manure  make 
it  probable  that  sometimes  an  increase  in  nitrogen  may  occur.  How- 
ever, these  are  rather  rare  exceptions ; usually  any  gain  that  may  take 
place  is  obliterated  by  large  losses  in  nitrogen. 

3.  PREVENTION  OF  LOSSES  OF  PLANT  FOOD  FROM  MANURES 

Because  the  biochemical  processes  taking  place  in  animal  manure 
are  incompletely  known,  it  is  impossible  at  the  present  time  to  decide 
definitely  how  the  great  losses  of  plant  food  can  be  prevented. 
But  it  is  certain  that  they  can  be  materially  reduced  if  the  neces- 
sary attention  and  care  is  given  to  this  problem,  and  if  proper  use  is 
made  of  the  methods  now  available.  The  benefit  which  may  be  derived 
from  such  practice  is  not  merely  a personal  matter  for  the  farmer, 
but  it  is  of  still  greater  importance  in  regard  to  the  conservation  of 
national  wealth.  A loss  of  several  hundred  million  dollars  -worth  of 
plant  food  every  year,  solely  because  of  improper  handling  of  manure, 
is  obviously  no  negligible  fact.  Mechanical,  chemical,  as  well  as  biological 
methods  may  be  used  to  reduce  these  losses.  The  first  ones  are  of  greatest 
importance,  while  the  second  and  the  third  are  playing  subsidiary  roles. 

Mechanical  Methods. — Separate  collection  of  solid  and  liquid 
manures  is  undoubtedly  the  most  effective  means  of  preventing  large 
losses  of  plant  food,  of  regulating  the  fermentative  processes  most 
advantageously,  and  of  attaining  the  best  possible  fertilizing  effects. 
Solid  excrements  and  straw  make  the  soil  richer  in  bacteria  and  in 
humus ; liquid  manure,  when  carefully  kept  in  air-tight  and  water-tight 
tanks,  contains  few  bacteria  but  comparatively  large  quantities  of 
available  nitrogen  and  potassium.  In  different  parts  of  continental 
Europe  this  method  of  handling  animal  manure  has  been  practiced 
successfully  for  a long  time.  Certain  practical  difficulties  exist, 
but  they  can  be  overcome,  and  they  are  greatly  surpassed,  as  a rule, 
by  the  advantages  of  this  method.  The  tanks  for  collecting  and  storing 
liquid  manure  must  be,  of  course,  water-tight  and  have  a well  fitting 
cover  that  prevents  the  escape  of  carbon  dioxide  and  of  ammonia.  The 
outlet  from  the  stable  should  not  be  above,  but  below  the  surface,  to 
prevent  disturbance  within  the  fermenting  liquid. 

If  feces,  straw,  and  urine  are  mixed,  the  manure  should  be  stored 
in  water-tight  covered  pits.  Fairly  satisfactory  results  are  assured 
if  the  manure  is  daily  brought  out  from  the  stable,  filled  up  in  strips 
6 to  8 feet  deep,  pressed  down,  moistened  with  water  if  necessary, 
and  kept  under  cover. 

Very  valuable  manure  can  also  be  secured  by  simply  leaving  it  in 


MICROORGANISMS  IN  BARNYARD  MANURES 


235 


the  stable  on  a water-tight  floor,  until  it  can  be  removed  to  the  field. 
Labor  and  expense  connected  with  the  preceding  methods  are  mostly 
saved;  and  because  the  desired  fermentative  processes  are  favored  by 
a tight  and  moist  condition  of  the  manure  pile,  a well  rotted  material 
of  comparatively  high  fertilizing  effect  will  result.  However,  hygienic 
reasons  are  adverse  to  this  arrangement;  the  shelter  of  the  farm 
animals  should  not  be  at  the  same  time  the  manure  pile.  The  air  is 
usually  bad  in  such  stables,  and  contagious  diseases,  such  as  tuber- 
culosis and  mastitis,  may  easily  be  carried  to  every  animal  in  the 
stable. 

Chemical  Methods. — Since  the  middle  of  last  century  many  tests 
have  been  made  to  find  a simple  and  efficient  chemical  method  for 
preventing  all  losses  of  plant  food  from  manure.  The  results  have 
been  disappointing,  because  the  fermentative  processes  are  far  too 
complicated  and  too  little  known  to  permit  of  an  intelligent  control 
by  chemical  means.  Gypsum,  rock  phosphates,  potassium  salts,  iron 
sulfate,  and  other  substances  have  been  found  to  be  of  little  or  no 
value.  Because  the  breaking  down  of  the  organic  substances  and  the 
multiplication  of  the  bacteria  is  desired,  these  processes  should  not 
be  disturbed  by  such  admixtures.  The  use  of  acid  phosphate  can  be 
recommended,  because  it  exerts  no  detrimental  effects,  fixes  part  of 
the  ammonia,  and  at  the  same  time  increases  the  phosphoric  acid  content 
which  is  always  low  in  animal  manure. 

If  the  liquid  manure  is  kept  separate,  practically  all  losses  of 
nitrogen  can  be  prevented  by  a careful  use  of  sulfuric  acid,  of  acid 
sodium  sulfate,  or  of  formaldehyd.  The  ammonium  carbonate  first 
formed  easily  breaks  up  to  carbon  dioxide  and  free  ammonia ; sulfuric 
acid  and  acid  sulfate  transform  it  to  the  non-volatile  ammonium  sul- 
fate, while  formaldehyd  changes  it  to  hexa-methylene-tetramin,  which 
is  readily  nitrified  in  the  soil.  In  careless  hands  concentrated  sulfuric 
acid  is  not  without  danger,  therefore,  acid  sodium  sulfate  deserves 
preference ; but  this,  as  well  as  formaldehyd,  should  not  be  added  before 
the  fermentation  of  the  urine  nitrogen  is  complete. 

Biological  Methods. — Since  an  atmosphere  of  carbon  dioxide  pre- 
vents the  disintegration  and  volatilization  of  ammonium  carbonate, 
tight  covers  are  to  be  recommended  for  the  conservation  of  solid  and 
liquid  manures.  It  has  also  proved  useful  to  leave  a small  quan- 
tity of  old,  well  fermented  material  in  the  pit  when  the  manure  is 
removed  to  the  field.  As  soon  as  fresh  material  is  added  to  the  old 
manure  a vigorous  fermentation  sets  in,  due  to  the  inoculation  taking 
place  from  the  old  to  the  new  manure,  and  the  desired  atmosphere  of 
carbon  dioxide  is  soon  restored. 


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If  large  quantities  of  whey,  molasses,  or  of  other  waste  products 
rich  in  soluble  carbohydrates  are  available,  they  may  be  added  to  the 
manure;  they  are  quickly  transformed  therein  to  lactic  and  other 
organic  acids,  which  contribute  to  the  fixation  of  ammonia.  A similar 
and  perhaps  more  efficient  biological  acid  production  in  manure  may 
possibly  be  effected  by  adding  commercial  sulfur  to  the  manure.  The 
sulfur  is  rapidly  oxidized  to  sulfuric  acid  which  is  neutralized  by  the 
ammonia.  Tests  made  at  the  Ohio  Experiment  Station  gave  very 
promising  results.  Manures  kept  for  250  days  without  and  with  sulfur 
showed  the  following  losses  d 


Percentage 

Untreated 

With  Sulfur 

Organic  substances 

32.5 

18.0 

N itrogen 

10.5 

3.5 

If  these  findings  are  confirmed,  and  if  the  cost  of  such  treatment  is 
not  too  high,  this  method  would  deserve  careful  consideration. 

1 J.  W.  Ames  and  T.  E.  Richmond,  Soil  Science,  vol.  4,  1917,  pp.  79-89. 


CHAPTER  XIV 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 

Physical,  chemical,  and  biological  factors  are  active  in  the  primary 
formation  of  soils  from  rocks,  as  well  as  in  the  secondary  transforma- 
tions continually  taking  place  in  all  soils.  Higher  and  lower  plants 
and  animals  participate  in  these  processes;  soil  bacteria  and  related 
microorganisms  constitute  a very  important  group  among  them.  Like 
all  living  organisms  they  are  influenced  by  the  physical  and  chemical 
conditions  prevailing  in  the  soils,  but  they  are  also  able  to  change 
these  conditions  to  a greater  or  less  extent.  Just  as  the  visible  plant 
growth  is  dependent  on  the  productivity  of  the  soil,  and  causes  at 
the  same  time  either  an  increase  or  a decrease  in  fertility,  so  the 
microflora,  too,  is  passively  and  actively  closely  connected  with  the 
special  condition  of  a soil.  Because  of  the  numerous  and  variable 
differences  in  the  structure  and  composition  of  the  soils  many  physical 
and  chemical  problems  are  still  to  be  solved,  and  in  soil  microbiology 
much  more  remains  to  be  done,  since  such  work  has  been  under  way 
only  during  forty  or  fifty  years.  Thus  far  a general  survey  has  been 
made,  and  the  more  important  subjects  have  been  studied.1 

1.  GERM  CONTENT  OF  SOILS 

Bacteria  are  of  greatest  importance  in  all  soils  in  regard  to  their 
number  as  well  as  to  their  activity.  Fungi  and  protozoa  are  usually 
less  frequent,  but  occasionally  they,  too,  may  become  quite  conspicuous. 
The  same  is  true  with  the  algae  in  soils.  Like  the  protozoa  they  do 
not  find,  as  a rule,  enough  moisture  in  field  soils,  for  their  requirements 
in  this  respect  are  generally  higher  than  those  of  the  bacteria,  and 
especially  of  the  lower  fungi.  In  addition  to  the  water  content,  the 
aeration  and  temperature  of  the  soil,  as  well  as  its  reaction  and  food 
supply,  are  the  main  factors  regulating  the  “life”  of  a soil.  Because 
these  conditions  are  continually  changing  in  every  soil,  it  is  quite 
obvious  that  the  microflora  of  a certain  soil  is  by  no  means  constant. 

1 For  detailed  references  see  F.  Lohnis,  “Handbuch  d.  landwirtschaftl.  Bak~ 
teriologie,”  Chap.  V,  and  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  54,  1921,  p.  285. 

237 


238 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


Season,  rainfall,  tillage,  manuring,  and  cropping  may  cause  far-reach- 
ing modifications.  Investigations  in  which  these  important  factors 
are  not  adequately  considered  are  of  very  little  value,  and  the  germ 
content  of  a soil  can  not  be  accurately  determined  by  one  or  a few 
simple  bacteriological  tests.  Such  studies  should  be  continued  for  at 
least  one  year. 

Frequency  of  Microorganisms. — Some  general  statements  about  the 
number  of  bacteria  and  of  other  microorganisms  present  in  soils  have 
been  made  on  p.  60.  There  it  was  also  pointed  out  that  these 
high  figures  should  not  be  overrated.  The  minute  size  of  the  bacteria 
and  their  uneven  distribution  in  the  soil  must  always  be  kept  in  mind. 
The  total  weight  of  the  soil  organisms,  however,  is  quite  remarkable 
and  clearly  indicates  that  far-reaching  physical  and  chemical  effects 
may  be  expected  from  such  a mass  of  living  matter. 

Bacteria  and  related  microorganisms  are  present  in  every  soil  from 
the  tropics  to  the  polar  regions,  and  even  desert  sands  and  alkali  soils 
which  are  almost  devoid  of  higher  plant  growth,  have  their  microflora. 
Of  course,  fertile  soils  are  more  thickly  populated  and  give  rise  to 
a much  more  varied  microflora,  but  even  in  such  soils  it  is,  as  a rule, 
only  the  upper  layer,  relatively  rich  in  humus,  that  offers  a most 
suitable  environment.  One  hundred  to  1000  million  of  bacteria,  fungi, 
algae,  and  protozoa  have  been  found  in  fertile  soil ; more  frequently 
5 to  50  millions  have  been  counted  per  g.  soil,  because  on  the  plate 
cultures,  which  are  used  as  the  basis  for  such  calculations,  only  part  of 
the  viable  microorganisms  will  produce  visible  colonies.  There  is  no 
possibility  of  inducing  all  bacteria  present  in  a soil  to  grow  on  any  one 
artificial  substrate,  and  all  results  obtained  are  therefore  merely  of 
relative  value.  They  have,  however,  very  clearly  shown  that  the 
frequency  of  microorganisms  rapidly  decreases  in  all  subsoils,  and  that 
this  decline  is  more  pronounced  in  the  heavy  and  humid  soils,  than  in 
the  light  and  arid  ones,  mainly  because  of  the  better  aeration  in  the 
latter  soils.  Higher  temperature  and  more  moisture  are  favorable  to 
a rapid  multiplication  of  the  bacteria  in  the  soil ; nevertheless,  a reduc- 
tion in  number  and  activity  is  frequently  noticeable  during  the  warmer 
season.  Although  other  factors  may  cause  modifications  of  this  rule, 
it  is  usually  during  spring  and  autumn  that  maximal  growth  and 
activity  occur  in  the  soils.  The  only  exception  is  fallow  soil,  where 
the  maxima  have  mostly  been  found  during  summer.  Frozen  soil 
often  gives  exceptionally  large  numbers  of  colonies  on  the  plates,  but 
this  is  merely  due  to  the  breaking  up  of  the  colonies  present  in  the 
soil  j1  the  situation  here  is  similar  to  that  found  in  centrifuged  milk. 

1 A.  F.  Vass,  Cornell  Agr.  Exp.  Stat.  Memoir  27,  1919. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


239 


Physiological  Groups  of  Soil  Organisms. — Among  the  organisms 
growing  in  plate  cultures  non-sporulating  rods  are  usually  most  fre- 
quent ; liquefying  and  non-liquefying  Fluorescent es,  B.  aerogenes, 
Proteus,  and  different  yellow  rods  are  found  quite  regularly.  Micrococci 
are  less  numerous,  and  also  the  sporulaiing  bacilli  are  not  as  frequent 
as  might  be  expected.  Actinomycetes  are  always  present;  especially 
those  producing  a soluble  dark  brown  pigment  are  very  characteristic 
on  soil  plates  (see  Fig.  1,  Plate  III).  Their  number  may  reach  50  per 
cent  of  the  total  figure  when  the  soils  tested  have  recently  received  an 
application  of  barnyard  manure  or  of  straw.  Fungi  predominate  in 
acid  soils,  especially  if  they  are  rich  in  humus ; forest  soils  show,  there- 
fore, a vigorous  mold  growth.  Protozoa  and  algae  are  rare  in  dry,  but 
frequent  in  wet  soils;  irrigation  causes  a strong  development  of  these 
organisms.  In  general  it  may  be  assumed  that  in  field  and  garden  soil 
for  every  1000  bacteria,  10  to  100  fungous  cells,  and  1 to  10  protozoa 
and  algae  may  be  present. 

The  microorganisms  growing  upon  the  substrates  commonly  used  for 
plate  cultures  in  the  laboratory  represent,  however,  only  a part  of  the 
microflora  of  the  soil,  and  very  important  physiological  groups  of  soil 
organisms,  such  as  the  nitrifying  and  nitrogen  fixing  bacteria  do  not 
grow  at  all,  or  only  to  a very  limited  extent  under  these  conditions. 
Special  tests  must  be  made  in  order  to  secure  information  upon  their 
distribution.  From  such  tests  it  was  found,  for  example,  that  a field 
soil  from  which  50  million  bacteria  per  g.  were  grown  on  the  gelatin 
plate,  showed  the  following  development  of  the  more  important  groups 
per  g.  soil,  if  this  was  used  for  inoculating  suitable  solutions  d 

75.000. 000  in  peptone  solution  162,500  denitrifying  bacteria 

25.000. 000  in  urea  solution  100,000  nitrifying  bacteria 

2,500,000  nitrogen  fixing  bacteria 

But  even  such  detailed  figures  are  not  of  great  value.  It  is  not  the 
number,  but  the  activity  of  the  soil  organisms  which  is  of  importance, 
and  the  latter  depends  on  the  former  to  a limited  extent  only.  High 
or  low  efficiency  of  the  individual  cells  determines  the  result  more  than 
does  the  number  and  the  species. 

Relation  Between  Germ  Content  and  Productivity  of  Soils. — In 

exceptional  cases  the  bacterial  number  obtained  by  plate  cultures  may 
display  a certain  parallelism  with  the  productivity  of  a given  soil. 
For  example,  number  and  productivity  are  low  in  distinctly  acid  soils, 
and  both  will  rise  as  soon  as  lime,  manure,  or  nitrate  are  applied ; but 
in  soils  of  normal  reaction  no  fixed  relations  exist  between  bacterial 
1 W.  Millard,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  31,  1911,  p.  502. 


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counts  and  crop  production.  The  indiscriminate  counting  of  soil  bac- 
teria is  indeed  no  better  than  it  would  be  to  enumerate  all  green  plants 
growing  on  an  acre  of  land,  quite  irrespective  of  the  kind  of  plant 
whether  weeds  or  cultivated  plants;  but  even  if  only  the  latter  were 
singled  out,  and  it  would  be  found,  for  instance,  that  there  are  two 
million  wheat  plants  on  a given  area,  such  a result  would  not  be  of 
great  interest  to  the  agriculturist.  Always  it  is  the  efficiency  which 
really  counts,  that  is,  the  crop  production  and  the  activities  of  the 
soil  organisms.  Naturally,  parallelisms  may  occur  between  number 
and  efficiency,  but  more  frequently  they  are  absent.  For  example, 
numbers  and  metabolic  effects  of  those  five  important  physiological 
groups  mentioned  above  were  determined  in  soil  samples  taken  from 
the  same  plots  in  January  and  in  July.  The  July  data  presented  below 
are  calculated  in  per  cent  of  those  obtained  in  January:1 


Ammonifi  cation 

From  Pepton 

From  Urea 

Nitri- 

fication 

Denitri- 

fication 

Nitrogen- 

fixation 

Numbers 

125 

100 

30 

100 

3000 

Effects 

113 

308 

77 

113 

80 

It  is  readily  admitted  that  all  such  figures  are  but  approximately 
correct,  because  no  method  is  known  that  would  furnish  quite  exact 
results ; but  from  all  what  is  known  concerning  the  widely  differing  and 
variable  efficiency  of  all  microorganisms,  it  is  practically  self-evident 
that  no  fixed  relations  can  be  expected  between  the  productivity  of 
soils  and  the  number  of  bacteria  living  therein. 

If  the  metabolic  effects  produced  by  the  soil  bacteria  are  determined 
under  different  conditions  much  more  valuable  conclusions  can  be 
drawn  concerning  the  productivity  of  the  soils  tested.  It  is  to  be  kept 
in  mind,  however,  that  the  activities  of  soil  organisms  are  greatly 
influenced  by  the  season;  they  are  generally  lowest  during  winter,  high 
in  spring  and  in  autumn,  and  again  lower  in  summer.  In  Fig.  55  a 
few  annual  curves  are  reproduced  which  show  very  clearly  these  varia- 
tions and  irregularities.2  Frequently  spring  and  autumn  maxima  have 
been  observed,  but  weather,  tillage,  liming,  manuring,  and  cropping 
may  cause  so  many  exceptions  to  this  rule  that  it  is  quite  indispensable 

1 F.  Lohnis,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  14,  1905,  p.  6. 

2 F.  Lohnis,  Mitteilungen  d.  Landwirtschaftl.  Instituts  d.  Univ.  Leipzig , Heft  7,  1905. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS  241 


to  extend  such  investigations  through  different  seasons,  and  never  to 
rely  upon  short-termed  and  isolated  observations. 

The  productivity  of  a soil  depends  on  its  chemical,  physical,  and 
biological  conditions.  The  first  ones  are  relatively  stable,  the  second 
ones  rather  variable,  and  the  third  ones  very  unstable.  Most  of  the 
potential  plant  food  in  the  soil  is  insoluble,  and  therefore  not  directly 
accessible  to  the  plant  roots;  but  at  the  same  time  it  is  protected 


against  being  lost  by  leaching.  Gradually  and  continually  rela- 
tively small  parts  of  this  large  stock  of  inert  material  are  being 
made  available,  mostly  by  the  direct  and  indirect  action  of  soil  organ- 
isms. The  transformation  of  carbonaceous  and  nitrogenous  substances 
is  almost  exclusively  the  work  of  bacteria  and  fungi,  while  the  dis- 
solution of  minerals  depends  in  the  first  place  on  the  degree  to  which 
the  soil  solution  is  saturated  with  carbon  dioxide,  which  again  is  largely 
produced  by  the  minute  inhabitants  of  the  soil.  Temperature,  mois- 


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ture,  tillage,  and  liming  are  of  greatest  influence  upon  the  physical 
conditions  of  a soil,  but  the  growth  and  incessant  activity  of  the  soil 
organisms  are  also  not  negligible  in  this  respect.  The  difference  in 
the  productivity  of  surface  soils  and  of  subsoils,  which  is  usually  most 
pronounced  in  heavy  soils  under  humid  conditions,  demonstrates  very 
clearly  the  eminent  importance  of  the  “life”  in  the  soil. 

Soil  Sickness. — Sometimes  it  happens  that  despite  heavy  applica- 
tions of  manure  and  fertilizers  the  productivity  of  a soil  shows  a 
marked  decrease,  especially  as  far  as  certain  crops,  such  as  clover, 
peas,  lupines,  flax,  cucumbers,  and  grapes  are  concerned.  It  is  cus- 
tomary to  speak  in  such  cases  of  clover  sickness,  etc.,  or  more  generally 
of  a sickness  or  a fatigue  of  the  soil.  Frequently  there  are  real  causes 
of  “sickness,”  that  is,  a multitude  of  disease  producing  organisms, 
fungi  as  well  as  insects,  have  accumulated  in  the  sick  soil,  because 
of  a too  often  repeated  planting  of  the  same  crop.  In  other  cases 
large  quantities  of  fertilizers  may  have  been  applied,  but  not  in  proper 
relation  to  each  other;  too  much  lime,  for  instance,  curtails  the  avail- 
ability of  potassium  for  certain  plants,  like  lupines  and  flax,  so  much 
that  they  may  become  “lime  sick.”  However,  even  if  all  chemical  as 
well  as  physical  conditions  are  well  taken  care  of,  as  in  greenhouses, 
and  no  damage  is  done  by  disease  producing 
organisms,  another  fatigue  of  the  soil  may  arise 
which  is  due  to  an  excessive  development  of  soil 
protozoa  that  prey  upon  the  soil  bacteria.  As  was 
said  before,  protozoa  are  favored  by  a high  mois- 
ture content  of  the  soil ; in  greenhouses  and  in 
irrigated  fields  the  situation  is  most  favorable  for 
them.  Bacteria  are  their  main  source  of  food,  and 
reciprocal  increases  and  decreases  of  protozoa, 
especially  of  amoebae,  and  of  bacteria  are  very 
conspicuous  under  such  conditions.1  Figure  56 
shows  two  such  animals  taken  from  a crude  cul- 
ture of  Azotobacter,  whose  large  globular  cells  have  been  in  part  in- 


Fig.  56. — Protozoa  in  a 
crude  culture  of  Azoto- 
bacter, living,  X1000. 


gested. 

However,  not  every  reduction  in  number  and  activity  of  the  useful 
soil  bacteria  can  be  explained  by  the  predatory  habit  of  soil  protozoa. 
A heavy  growth  of  cultivated  plants  may  also  cause  such  a reduction, 
either  by  the  lowering  of  the  moisture  content  of  the  soil,  or  by  the 
production  of  a considerable  amount  of  organic  substances  that  act 
unfavorably  upon  many  soil  bacteria,  as  well  as  upon  higher  plants. 


-D.  W.  Cutler  and  L.  M.  Crump,  Annals  of  Applied  Biology,  vol.  /,  1920,  p.  11. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS  243 


It  is  an  old  practical  experience  that  many  soils  need  a rest  after 
a crop  was  taken,  in  order  to  regain  their  normal  productivity,  and  it 
has  also  repeatedly  been  ascertained  that  the  biological  activities  in 
the  soil  have  the  tendency  to  decline  during  the  summer  in  cropped 
fields,  while  they  do  not  decrease  in  fallow.  That  this  is  partly  due  to 
the  presence  of  soluble  organic  substances,  which  can  be  removed  by 
leaching,  has  been  shown  experimentally,1  and  very  probably  soil  sick- 
ness is  also  caused  to  some  extent  by  such  accumulations  which  under 
ordinary  conditions  are  decomposed  by  soil  organisms  before  the  next 
crop  is  planted. 

Biological  Soil  Tests. — It  is  a comparatively  simple  matter  to  make 
plate  cultures,  to  show  the  presence  of  microorganisms  in  a soil,  or 
to  add  different  substances  to  samples  of  soil,  to  keep  them  for  a few 
weeks,  and  then  to  prove  by  chemical  analysis  what  transformations 
have  taken  place  in  these  samples  as  compared  with  others  that  had 
not  been  treated  or  had  been  sterilized  before.  However,  it  should 
never  be  overlooked  that  all  experiments  in  the  laboratory  are  made 
under  conditions  which  are  very  different  from  those  regulating  the 
life  of  the  soil  organisms  in  the  field.  Therefore,  in  drawing  any 
conclusions  or  basing  any  generalizations  upon  such  results  very  great 
care  should  be  exercised.  It  has  been  emphasized  that  because  of  the 
instability  of  bacterial  activities  all  short-timed  biological  soil  tests 
are  of  little  value ; furthermore  it  should  always  be  kept  in  mind  that 
because  laboratory  tests  are  invariably  conducted  under  conditions 
differing  from  those  in  the  greenhouse  or  in  the  field,  they  should  be 
coordinated  as  far  as  possible  with  pot  and  plot  experiments,  whose 
results  will  help  to  prevent  incorrect  generalizations  and  will  materially 
increase  the  value  of  such  tests. 

For  preparing  solid  and  liquid  substrates  for  soil  organisms  an 
extract  made  by  heating  soil  with  an  equal  amount  of  water,  has 
proved  very  useful.  The  chemical  qualities  of  the  particular  soil  to 
be  tested  are  thus  in  part  retained  in  the  bacteriological  test,  and  very 
frequently  soil  extract  has  been  found  to  be  a much  better  substrate 
than  any  artificial  solution,  because  too  little  is  known  about  the  real 
composition  of  the  soil  solution,  as  well  as  about  the  most  suitable 
growth  conditions  of  the  various  groups  of  soil  organisms.  Meat  sub- 
strates and  blood  serum  are  usually  best  for  the  cultivation  of  the 
bacteria  living  in  man  and  animal;  solutions  prepared  from  pure 
chemicals  dissolved  in  distilled  water  are  usually  much  inferior.  With 
soil  organisms  the  situation  is  very  similar. 

'A.  LuMifiRE,  Cornet,  rend.  Acad.  Paris,  vol.  171,  1920,  p.  868. 


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Occasionally  the  opinion  has  been  expressed  that  the  activities  of 
soil  organisms  could  be  studied  accurately  only  in  the  soil  itself,  and 
that  any  solution,  made  either  from  soil  extract  or  from  pure  chemicals, 
be  unsuitable.  However,  careful  consideration  of  what  is  known  at 
present  about  bacteria  shows  very  clearly  that  almost  every  discovery 
since  the  time  of  Leeuwenhoek,  including  all  information  abuut  am- 
monifying, nitrifying,  denitrifying,  nitrogen  assimilating,  and  other 
organisms,  has  been  derived  from  cultivating  these  organisms  in 
properly  prepared  solutions.1  In  the  soil  itself  the  bacteria  live  in  the 
solution  circulating  around  and  between  the  solid  particles.  Un- 
doubtedly, tests  made  in  soil  are  of  value,  too,  but  because  of  the 
complex  and  largely  unknown  composition  of  this  substrate  the  results 
obtained  leave  much  to  be  desired.2  In  all  such  tests  the  chemical 
and  physical  conditions  in  the  soil  samples  are  so  different  from  those 
prevailing  in  the  greenhouse  or  in  the  field  that  again  no  rash  con- 
clusions or  generalizations  would  be  permissible.  If  the  soil  is  dried 
before  being  used,  and  excessive  quantities  of  the  substances  to  be 
tested  are  added  to  it,  misleading  results  are  almost  inevitable.3 

2.  BACTERIAL  ACTIVITIES  IN  SOIL 

In  the  soil  all  organic  residues  are  mineralized  by  the  activities 
of  bacteria  and  related  microorganisms.  Carbonaceous  and  nitrogenous 
compounds  of  almost  endless  variety  are  being  decomposed,  and  many 
intermediary  products  are  formed  that  at  the  present  time  are  not 
well  known.  Especially  concerning  the  dark  colored  soil  constituents, 
collectively  called  humus,  comparatively  little  has  been  ascertained. 
Much  more  complete  data  are  available  in  regard  to  the  metabolism 
of  nitrogen ; the  relative  scarcity  of  this  element  in  agricultural  soils, 
its  high  economic  value,  and  its  evasive  nature  have  necessarily  at- 
tracted the  attention  of  all  investigators.  Since  the  nitrogen  trans- 
formations are  largely  influenced  by  the  quantity  and  quality  of  the 
carbonaceous  substances  present  in  the  soil,  the  metabolism  of  the 
latter  is  also  of  great  interest.  These  problems  have  been  discussed 
in  their  general  aspects  in  Chapter  VII,  2-4;  on  the  following  pages 

1 M . W.  Beijerinck,  Jaarboek  d.  Akad.  Amsterdam,  1913. 

2 F.  Lohnis  and  H.  H.  Green,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  37,  1913,  p.  534; 
vol.  40,  1914,  p.  457. 

3 W.  P.  Kelley,  Jour.  Agr.  Research,  vol.  7,  1916,  p.  417.  Further  details  upon 
biological  soil  tests  are  given  in  the  authors’  laboratory  manuals  mentioned  on  pp.  11 
and  12.  In  regard  to  experiments  on  soil  protozoa  see  N.  Kopeloff,  H.  C.  Lint  and 
D.  A.  Coleman,  Centralbl.  f.  Bakt.,  II.  Abt.,  vol.  45,  1916,  p.  230;  and  D.  W.  Cutler, 
Jour.  Agr.  Science,  vol.  10,  1920,  p.  135. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


245 


they  will  be  considered  in  their  relations  to  soil  fertility.  In  the  trans- 
formations of  mineral  substances,  such  as  phosphates,  potassium  salts, 
etc.,  bacterial  activities  are,  as  a rule,  of  secondary  importance,  as  has 
been  explained  in  Chapter  VII,  5. 

Carbon  Metabolism. — The  quantities  of  organic  substances  which 
remain  in  the  fields  or  are  returned  to  them  in  the  form  of  organic 
manures,  are  quite  considerable.  The  dry  weights  of  crop  residues, 
that  is,  of  stubble  and  roots,  vary  according  to  the  intensity  of  cul- 
tivation usually  between  800  and  3000  lbs.  per  acre.  An  average 
dressing  with  stable  manure  furnishes  approximately  6000  to  8000  lbs. 
of  organic  substances,  while  in  the  form  of  green  manures  2000  to 
10,000  lbs.  may  be  added,  dependent  on  the  growth  of  these  plants. 
The  total  dry  weight  of  carbonaceous  substances  returned  annually 
to  the  soil,  varies  according  to  the  type  of  farming,  between  1000  and 
6000  lbs.  per  acre ; 3000  lbs.  may  be  accepted  as  a moderate  average 
for  fields  receiving  organic  manures  regularly,  though  not  abundantly. 

Carbon  dioxide  and  humus  are  the  main  products  formed  by  bac- 
terial action  from  all  these  organic  residues.  Organic  acids,  alcohols, 
methane,  and  hydrogen  may  appear  as  by-products,  but  the  last-named 
gases  are  practically  absent  in  well-aerated  soils,  while  they  are 
regularly  present  in  swampy  lands,  wherein  the  organic  acids  are  also 
more  frequent.  Strong  aeration  and  high  temperatures  favor  the 
formation  of  carbon  dioxide,  while  under  the  opposite  conditions  large 
quantities  of  very  resistant  humus  are  produced.  Both  extremes  are 
detrimental  to  the  fertility  of  the  soil,  which  is  best  maintained  if  a 
moderate  amount  of  neutral  humus  is  always  retained  and  rebuilt  in 
the  soil,  as  is  done  in  garden  land  and  in  highly  productive  fields. 

Formation  of  Carbon  Dioxide. — If  annually  3000  lbs.  of  organic 
matter  are  returned  per  acre,  and  the  humus  content  of  the  land  is 
sufficiently  high,  so  that  these  organic  substances  can  be  oxidized  to 
carbon  dioxide  without  depleting  the  soil,  approximately  6000  lbs.  of 
this  gas  will  be  produced,  if  50  per  cent  is  accepted  as  the  average 
carbon  content  of  the  organic  substances.  The  volume  of  these  6000 
lbs.  of  carbon  dioxide  is  approximately  1600  m.3 ; if  all  this  gas  would 
be  produced  simultaneously  and  could  be  kept  together,  one  acre  of 
land  would  be  covered  by  a uniform  layer  of  carbon  dioxide  16  inches 
deep.  It  is  obvious  that  these  large  quantities  of  carbon  dioxide  must 
be  of  great  influence  upon  the  crop  production.  On  the  one  side  the 
green  plants  are  supplied  with  carbon  dioxide  for  their  assimilation ; 
on  the  other  side  the  mineral  constituents  of  the  soil  are  attacked 
by  the  carbon  dioxide  dissolved  in  the  soil  solution,  and  thus  inert 
plant  food  is  made  soluble.  The  roots  of  the  cultivated  plants  also 


246 


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produce  carbon  dioxide  and  participate  in  the  dissolution  of  the 
minerals  in  the  soil,  but  the  quantities  of  carbon  dioxide  produced  by 
the  soil  organisms  are,  as  a rule,  much  larger,  provided  enough  organic 
substances  are  present  in  the  soil,  or  are  regularly  returned  to  it.1 

After  a heavy  application  of  barnyard  or  green  manure  the  produc- 
tion of  carbon  dioxide  is  sometimes  so  vigorous  that  the  soil  atmosphere 
temporarily  loses  all  of  its  oxygen.  Ten  per  cent  of  carbon  dioxide 
have  been  found  frequently,  but  the  lowered  oxygen  tension  is  still 
sufficient  for  a normal  nitrification  and  at  the  same  time  most  favorable 
for  the  formation  and  conservation  of  humus  in  the  soil.  Since  high 
soil  temperatures  and  ample,  though  not  excessive,  soil  moisture 
stimulate  the  respiration  of  the  soil  organisms,  the  resulting  increase 
and  decrease  of  carbon  dioxide  production  may  be  used  as  a fairly 
reliable  measurement  of  the  bacterial  activity  of  a soil.  Quite  exact 
results,  however,  are  not  obtained,  because  the  removal  of  the  soil 
samples  from  the  field  changes  the  physical  conditions  to  a smaller 
or  larger  extent,  dependent  on  the  manner  in  which  the  samples  are 
taken  and  handled.  Small  quantities  of  carbon  dioxide  may  be  pro- 
duced by  sterilized  soil,  too,  but  they  are  practically  negligible. 

Decomposition  of  Different  Carbon  Compounds. — Part  of  the  or- 
ganic substances,  for  instance,  straw,  corn  stalks,  stubble  and  roots, 
solid  excrements,  and  peat  litter  are  decomposed  slowly ; they  produce 
more  humus  than  carbon  dioxide.  Other  substances,  such  as  young 
green  plants,  are  quickly  disintegrated,  and  most  of  their  carbon  is 
oxidized  to  carbon  dioxide;  green  manure  plowed  under  during  summer 
vanishes  often  entirely  from  the  soil  before  the  next  crop  is  planted. 
The  following  figures  show  how  much  of  the  carbon  content  of  various 
substances  was  oxidized  within  3 weeks,  if  these  were  added  in  every 
case  at  the  rate  of  10  g.  carbon  to  500  g.  of  soil.2 


Per  Cent 


Red  clover 59.69 

Glucose 42  14 

Starch 29.00 


Per  Cent 


Oak  leaves 17.70 

Wheat  straw 14.54 

Cellulose 11.77 


Because  of  the  comparatively  large  quantities  of  carbon  added  to  the 
soil  in  this  experiment,  the  data  obtained  are  merely  of  relative  value. 
In  the  field  much  smaller  amounts  are  applied,  and  the  transformation 
is  more  rapid.  For  example,  in  laboratory  experiments  where  0.5  per 
cent  of  cellulose  was  mixed  with  soil,  70  to  100  per  cent  of  it  was 


1 E.  B.  Fred  and  A.  R.  C.  Haas,  Jour.  General  Physiol.,  vol.  1,  1919,  p.  631. 

2 J.  Dvorak,  Zeitschr.f.  d.  landw.  Fers.  Wesen  in  Oesterreich,  vol.  15,  1912,  p.  1097. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


247 


decomposed  within  one  month.1  Under  field  conditions  usually  not 
more  than  0.1  per  cent  of  the  soil  weight  is  added  annually  in  the 
form  of  organic  residues,  but  because  all  transformations  proceed  more 
slowly  in  the  field  than  in  the  laboratory,  these  relatively  small  quan- 
tities suffice,  as  a rule,  to  maintain  the  carbon  balance  in  the  soil. 

The  decomposition  of  cellulose  in  field  soils  is  usually  done  by 
aerobic  bacteria  and  fungi.  If  sheets  of  paper  are  placed  on  top  and 
8 to  10  in.  below  the  surface  in  soil  whose  water  holding  capacity  is 
fully  saturated,  after  2 to  3 weeks  the  decomposition  is  very  marked 
in  the  first,  but  hardly  noticeable  in  the  second  case.  Figure  57  illus- 
trates this  fact.  Because  a very  large  number  of  soil  fungi  are  capable 
of  dissolving  cellulose,  their  growth  is  greatly  enhanced  if  this  sub- 
stance is  added  to  the  soil.  It  has  been  observed,  for  instance,  that 


Fig.  57. — Cellulose  decomposition  in  soil  (a)  at  the  surface,  ( b ) 8 in.  below  the  surface. 

under  such  conditions  their  number  rose  from  100,000  to  200,000,000 
per  g.  soil.2 

Formation  of  Humus. — A larger  or  smaller  part  of  the  organic 
residues  incorporated  in  the  soil  is  transformed  into  humus.  If  no 
cultivation  takes  place,  as  in  grassland  and  forests,  the  humus  content 
shows  a gradual  and  continuous  increase,  and  if  this  process  extends 
through  long  periods  as  in  virgin  soils,  enormous  quantities  of  humus 
are  accumulated  which  constitute  an  extremely  valuable  store  of 
potential  plant  food.  Unfortunately,  after  such  soils  have  been  taken 
under  cultivation  their  original  fertility  has  frequently  been  wasted, 
and  lack  of  humus  is  now  often  the  main  cause  of  the  reduced  pro- 
ductivity of  such  land. 

1 C.  Mutterlein,  Studien  xiber  die  Zersetzung  der  Zellulose,  Diss.  Leipzig,  1913,  p.  96. 

2 F.  M.  Scales,  Botan.  Gazette,  vol.  60,  1915,  p.  149. 


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Because  practically  all  nitrogenous  as  well  as  non-nitrogenous 
organic  substances  can  be  changed  to  humus,  the  chemical  composi- 
tion of  the  latter  varies  in  all  soils.  Carbohydrates,  especially  cellulose, 
are  usually  completely  decomposed  if  fully  aerobic  or  strictly  anaerobic 
conditions  favor  the  action  of  bacteria.  Fungi  and  actinomyeetes,  on 
the  other  hand,  are  more  inclined  to  produce  dark  humus-like  bodies 
when  growing  on  cellulose  (see  Fig.  2,  Plate  VIII). 

Much  of  the  finely  divided,  rich  humus  of  field  and  garden  soil 
is  made  up  of  the  excrements  of  worms  and  insects  which  constitute 
an  important  part  of  the  soil  fauna.  They  all  feed  on  the  organic 
residues  upon  and  within  the  soil,  and  this  transformation  of  plant 
substance  into  animal  excrements  plays  by  no  means  a subordinate 
role.  Counts  made  at  the  Rothamsted  Experimental  Station  showed 
that  per  acre  4 to  10  millions  of  insects,  worms,  and  myriapods  were 
present  in  field  soil ; manuring  caused  a marked  increase  in  their 
number.1 

Decomposition  of  Humus. — If  no  organic  manures  are  used,  and 
only  the  crop  residues  are  left  on  the  field,  sooner  or  later  the  soil 
will  be  robbed  of  most  of  its  humus  and  of  its  natural  fertility.  In- 
tensive clean  cultivation,  as  practiced  in  corn  fields  and  orchards, 
together  with  high  summer  temperatures  accelerate  this  process  un- 
avoidably ; accordingly,  vast  stretches  of  American  soil  have  lost  much 
or  nearly  all  of  their  original  humus  content.  Under  average  condi- 
tions 4 to  5 per  cent  of  the  humus  are  decomposed  annually;  that  is, 
in  a soil  with  2 per  cent  humus  approximately  3000  to  4000  lbs.  per 
acre.  From  what  was  said  above  concerning  the  carbon  metabolism 
it  is  quite  evident  that  these  losses  of  humus  necessitate  the  intelligent 
use  of  barnyard  and  green  manures. 

The  decomposition  of  the  carbon  in  humus  proceeds  generally  more 
rapidly  than  that  of  its  nitrogen;  old  humus  is  therefore,  as  a rule, 
richer  in  nitrogen  than  is  that  of  more  recent  origin.  Under  average 
conditions  1 to  2 per  cent  are  nitrified  annually;  in  soil  containing 
0.1  per  cent  of  nitrogen  this  is  equivalent  to  40  to  80  lbs.  of  nitrogen 
per  acre.  In  prairie  land  the  nitrogen  content  is  frequently  consid- 
erably higher,  and  often  much  more  humus  nitrogen  is  nitrified  than 
can  be  used  by  the  crops,  which  results  in  great  losses  by  leaching. 
Humus  of  different  origin  may  behave  very  differently  in  regard  to 
the  nitrification  of  its  nitrogen.2  Stable  manure,  green  manure,  peat, 
and  straw  were  kept  in  clean  sand  for  4^2  months ; the  humus  formed 

1 Rothamsted  Exp.  Stat.  Report  1918-20,  p.  20. 

2F.  Lohnis  and  H.  H.  Green,  Centralbl.  f . Bakt.,  II.  Abt.,  vol.  40,  1914,  p.  56. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


249 


was  then  extracted  and  mixed  with  soil.  After  5 weeks  the  nitrate 
nitrogen  was  determined  and  calculated  in  per  cent  of  the  total 
nitrogen : 


Humus  from 

Percentage 

Stable  Manure 

Green  Manure 

Peat 

Straw 

Total  nitrogen 

3.53-3.67 

4.16 

2.29 

1.34 

Nitrate  nitrogen 

11.2  -16.3 

14 -IS 

2. 9-3. 8 

0 

The  great  resistance  of  peat  nitrogen  is  very  marked.  The  straw  humus 
was  not  nitrified  at  all,  and  it  even  caused  a reduction  in  the  nitrate 
content  of  the  soil,  although  not  to  the  same  extent  as  is  characteristic 
of  fresh  straw. 

Soil  Acidity. — Unless  neutralized  by  lime,  most  of  the  humus  is  of 
distinctly  acid  reaction,  and  because  many  of  the  processes  connected 
with  the  transformation  and  mineralization  of  organic  residues  lead 
to  the  formation  of  acids,  there  is  a natural  tendency  at  least  in  humid 
soils  not  naturally  rich  in  lime  to  become  more  or  less  acid.  Absorptive 
processes  and  mineral  acids  contribute  to  the  soil  acidity,  but  wherever 
humic  acids  are  present  in  considerable  quantities  they  always  play  a 
prominent  role.  If  they  are  not  neutralized,  the  humus  decomposition 
remains  very  low,  because  of  the  inactivity  of  the  majority  of  soil 
organisms.  As  soon  as  the  reaction  is  adjusted  by  liming,  a vigorous 
bacterial  activity  sets  in  even  in  peat  soils,  and  excessive  liming  may 
sometimes  become  detrimental  because  of  too  rapid  decomposition  of 
the  humus  and  great  losses  in  nitrogen. 

Tilth  and  Ripening  of  the  Soil. — Formation  and  decomposition  of 
humus  play  a very  important  role  in  securing  the  desired  tilth  of  the 
soil.  This  particular  condition  which  is  essential  for  obtaining  regularly 
large  and  healthy  crops,  can  not  be  established  merely  by  careful 
tillage  of  the  soil.  Good  tillage  is  very  necessary,  but  the  best  possible 
physical  condition  of  the  soil  is  not  secured  by  mechanical  treatment 
alone.  If  the  soil  is  covered  with  a thick  layer  of  organic  residues, 
as  in  virgin  woodland  and  in  mulched  orchards,  it  will  often  exhibit 
a physical  structure  superior  to  that  of  cultivated  land.  Such  soil 
is  not  loose,  but  of  a peculiar,  crumbly  and  elastic,  mellow  texture, 
which  is  due  to  colloid  reactions  as  well  as  to  the  direct,  action  of  all 
the  worms  and  insects  that  participate  in  the  formation  and  trans- 
formation of  the  humus.  The  moisture  content  of  covered  soils  is 


250 


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higher  and  more  evenly  distributed  than  in  cultivated  land,  and  the 
chemical  and  biochemical  processes  are  therefore,  as  a rule,  greatly 
stimulated.  Noxious  organic  substances  are  rapidly  decomposed,  inert 
plant  food  is  made  soluble,  and  perfect  tilth  is  ultimately  established 
which  comprises  the  best  possible  physical,  chemical,  and  biological 
conditions  of  the  soil,  and  thereby  furnishes  a reliable  basis  for  secur- 
ing large  and  healthy  crops. 

It  always  takes  time  before  this  aim  is  reached.  If  the  climatic 
conditions  are  favorable,  if  tillage  and  manuring  are  done  efficiently, 
if  the  soil  reaction  is  right  and  the  humus  content  sufficiently  high, 
the  desired  tilth  can  be  attained  within  2 to  3 months.  If  the  climatic 
conditions  are  unfavorable,  if  the  physical  structure  of  the  soil  is  bad 
and  its  biological  activity  low,  a longer  time  is  needed,  and  in  extreme 
cases,  when  the  crop  production  becomes  entirely  unsatisfactory,  the 
field  must  be  kept  and  treated  as  fallow  for  a full  year. 

A soil  which  is  not  in  good  tilth  is  properly  termed  “raw”;  if  it  is 
planted  the  crop  will  be  comparatively  low.  Soil  in  good  tilth  on  the 
other  hand  may  be  called  “ripe,”  because  it  is  ready  to  produce  a 
good  crop,  and  the  slow  processes  that  lead  to  this  stage  constitute 
in  their  entirety  a real  “ripening”  of  the  soil.  In  the  European  agri- 
cultural literature  the  problem  of  how  to  obtain  a perfect  tilth  of  the 
soil  has  been  discussed  extensively,  and  the  term  tilth  (in  German 
“Gare”)  has  frequently  been  interpreted  as  meaning  “fermentation.” 
Because  carbon  dioxide  is  produced  and  the  structure  of  the  soil  be- 
comes soft  and  elastic,  the  tilth  of  the  soil  has  been  compared  with 
the  leavening  of  bread;  but,  the  two  processes  are,  in  fact,  rather  dif- 
ferent. It  is  not  alone  a fermentation,  but  a general  mellowing  or 
ripening  of  the  soil  that  takes  place. 

Nitrogen  Metabolism. — Comparatively  little  plant  food  enters  the 
soil  in  directly  available  form ; nitrate,  acid  phosphate,  and  potassium 
salts  are  fertilizers  which  are  immediately  accessible  to  the  plant  roots. 
Nearly  all  other  fertilizers  and  manures,  as  well  as  all  crop  residues, 
must  first  be  transformed  by  bacterial  action.  When  incorporated  in 
the  soil,  they  represent  sources  of  food  and  energy  for  the  minute 
inhabitants  of  the  soil,  and  only  the  metabolic  products  of  the  micro- 
organisms are  taken  up  by  the  cultivated  plants.  This  indirectly 
fertilizing  effect  of  all  organic  substances,  and  especially  of  all  organic 
nitrogenous  compounds  is  the  reason  why  the  nitrogen  given  in  this 
form  never  produces  such  prompt  and  uniform  results  as  are  obtained 
by  the  application  of  nitrate.  The  more  complex  the  nitrogenous  com- 
pounds are,  as  in  barnyard  manure,  green  manure,  horn  meal,  and 
bone  meal,  the  more  stages  of  the  nitrogen  cycle  are  to  be  passed  before 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


251 


nitrate  is  produced;  and  the  more  opportunities  are  offered  to  the  soil 
organisms  to  use  the  nitrogen  for  their  own  purposes,  the  less  nitrogen 
becomes  available  for  final  nitrification.  It  is  of  great  practical  im- 
portance to  find  out  under  what  conditions  these  transformations 
proceed  in  the  soil  in  such  a manner  that  the  most  satisfactory  fer- 
tilizing effect  will  be  realized,  but  not  very  much  is  known  about  this 
subject,  and  the  major  part  of  this  work  remains  to  be  done. 

Formation  of  Ammonia  from  Farm  Manures. — It  was  pointed  out 
in  the  preceding  chapter  that  the  mineralization  of  the  nitrogen  in 
barnyard  manure  is  almost  completely  checked  if  the  material  is 
plowed  under  before  the  carbonaceous  substances  are  partly  decom- 
posed, and  it  was  also  emphasized  that  a prompt  fertilizing  effect  is 
to  be  expected  only  from  the  liquids.  These  important  facts  are  further 
illustrated  by  the  following  results  obtained  in  nitrification  tests  made 
with  manure  of  different  age.1  The  materials  were  mixed  with  soil 
and  the  increase  in  mineral  nitrogen  (ammonia  and  nitrate)  was  deter- 
mined after  6 weeks  and  calculated  in  per  cent  of  the  total  nitrogen 
added  in  the  manure  : 


Manure  Used 

Percentage  of  Nitrogen 

Fresh 

1 Wk.  Old 

2 Wks.  Old 

4 Wks.  Old 

f straw  and  feces 

Mineralized  from  < , r . 

1 straw,  feces,  and  urine 

2 

6 

21 

31 

7 

31 

58 

75 

Because  in  the  field  the  conditions  for  bacterial  action  are  much 
less  favorable  than  in  the  small  soil  samples  used  in  the  laboratory, 
these  data  are  again  of  merely  relative  value.  Under  field  conditions 
rarely  more  than  10  to  20  per  cent  of  the  nitrogen  in  straw  and  feces 
are  mineralized  within  the  first  year,  whereas  30  to  50  per  cent  may 
become  available  if  liquids  and  solids  had  been  mixed,  and  60  to  80 
per  cent  if  the  liquid  manure  was  applied  separately.  Usually  the 
transformation  of  the  urine  nitrogen  to  ammonia  is  complete  before 
the  liquid  manure  is  brought  out  to  the  field.  If  the  ammonia  has  not 
been  fixed  by  sulfuric  acid  or  by  formaldehyde,  as  was  recommended 
on  p.  235,  large  quantities  of  it  may  be  lost  when  the  liquid  manure  is 
spread  and  left  on  the  surface. 

With  green  manures  similar  differences  in  the  mineralization  of 
nitrogen  may  be  observed.  Variations  of  the  fertilizing  effect  between 

1 F.  Lohnis  and  J.  H.  Smith,  Fiihling’s  landw.  Zeitg.,  vol.  63,  1914,  p.  153. 


252 


TEXTBOOK  OF  AGRICULTURAL  BACTERIOLOGY 


15  and  85  per  cent  are  not  infrequent.  As  a rule,  the  nitrogen  of 
young  plants  is  more  easily  mineralized  than  that  of  older  plants, 
because  more  of  it  is  present  in  the  form  of  amino  nitrogen,  and  the 
nitrogen-carbon  ratio  is  more  favorable  to  the  ammonifying  bacteria. 
If  old  material  rich  in  carbonaceous  substances  is  used,  a partial  rotting 
is  as  advisable  as  it  is  with  stable  manure.  If  circumstances  permit, 
it  is  better  to  leave  such  green  manure  over  winter  on  top  of  the  soil 
than  to  plow  it  under  in  fall.  In  China  and  in  India  special  rotting 
processes  have  long  been  practiced  in  connection  with  green  manuring. 
Neither  barnyard  nor  green  manure  should  be  buried  deep  into  the  soil, 
because  there  the  mineralization  would  be  very  incomplete.  On  the 
other  hand,  the  decomposition  will  usually  be  too  rapid,  if  young  green 
plants  are  turned  under  during  the  hot  season. 

Formation  of  Ammonia  from  Organic  Fertilizers. — Commercial  or- 
ganic fertilizers,  such  as  dried  blood,  fish  guano,  horn  meal,  rape  cake, 
and  cyanamid,  are  also  known  to  exert  rather  variable  effects,  because 
their  nitrogen  again  must  first  be  mineralized  by  soil  organisms.  If 
more  laboratory  tests  would  be  made  concerning  the  ammonification 
and  nitrification  of  these  fertilizers  in  different  types  of  soil  and  under 
different  climatic  conditions,  and  if  such  tests  would  be  connected 
with  field  experiments,  the  real  value  of  the  various  organic  fertilizers 
would  be  undoubtedly  much  better  known  than  it  is  at  present.  The 
curves  reproduced  on  p.  241,  showed,  for  example,  that  in  May,  that  is, 
at  the  time  when  most  of  the  nitrogen  was  made  available,  in  laboratory 
tests  25  per  cent  of  the  bone  meal  nitrogen  and  65  per  cent  of  the 
cyanamid  nitrogen  were  ammonified.  The  percentage  of  fertilizer 
nitrogen  taken  up  by  the  potato  crop  grown  on  this  land  proved  to  be 
25.3  and  61  per  cent  respectively.  Cyanamid  and  urea  are  undoubtedly 
the  most  valuable  among  the  commercial  organic  nitrogen  fertilizers. 
Cyanamid  itself  is  poisonous  to  green  plants  and  should,  therefore,  be 
applied  several  weeks  before  planting.  Soil  colloids  effect  a rapid 
transformation  of  cyanamid  into  urea,  and  the  urea  is  changed  to 
ammonium  carbonate  by  bacterial  action.  Various  soil  fungi  are 
capable  of  attacking  cyanamid  directly,  but  the  rate  of  ammonification 
is  rather  low.  Because  of  the  much  more  rapid  action  of  the  soil 
colloids  and  the  fairly  complete  ammonification  which  was  regularly 
observed  in  laboratory  tests,  the  first-named  process  is  undoubtedly  of 
greater  importance.  As  a rule,  non-sporulating  bacteria  are  most  fre- 
quent in  ammonification  tests,  but  sporulating  bacilli,  though  less 
numerous,  are  often  much  more  efficient.  Ammonifying  fungi  dominate 
in  acid  soils;  in  neutral  soils  they  are  less  conspicuous,  though  not 
absent. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


253 


Nitrification. — Many  experiments  have  demonstrated  that  all  green 
plants  may  assimilate  ammonia  nitrogen,  and  certain  plants,  such  as 
rice,  grow  even  better  with  ammonia  than  with  nitrate;  but  the  ma- 
jority of  cultivated  plants  prefer  the  nitrate  nitrogen.  Prompt  nitri- 
fication of  ammonia  nitrogen  is  generally  favorable,  because  ammonium 
carbonate  may  exert  a caustic  effect  upon  the  plant  roots,  resulting 
in  the  “burning”  of  the  plants,  which  is  sometimes  observed  when 
liquid  manure  is  brought  in  contact  with  growing  crops.  Such  damage 
would  be  prevented  only  in  acid  soils  and  in  those  of  very  high  ab- 
sorptive power;  in  all  other  soils  nitrification  is  distinctly  useful. 

Although  the  nitrifying  bacteria  prefer  a slightly  alkaline  reaction, 
the  average  hydrogen-ion  concentration  in  soils,  which  is  usually  some- 
what below  pH  = 7,  is  still  sufficient,  and  nitrification  has  been 
observed  even  in  distinctly  acid  peat  and  forest  soils.  In  acid  soils 
the  transformation  is  likely  to  proceed  in  a somewhat  abnormal  manner 
insofar  as  unusually  large  quantities  of  nitrite  may  accumulate,  which 
may  prove  injurious  to  the  cultivated  plants.  In  normal  soils  hardly 
any  nitrite  can  be  found ; both  groups  of  nitrifying  bacteria  cooperate 
closely,  and  the  ammonia  nitrogen  is  oxidized  to  nitrate  without  any 
considerable  loss.  Two  French  agricultural  chemists,  Th.  Schlosing, 
sr.  and  jr.,  have  made  interesting  experiments  upon  this  problem, 
which  furnish  a clear  picture  of  the  influence  exerted  upon  nitrification 
by  the  composition  and  moisture  content  of  the  soil.1  Sand  and  clay 
were  mixed  in  different  proportions,  and  so  much  water  was  added 
that  in  all  cases  it  was  equal  to  9.5  per  cent  of  the  soil  weight.  Within 
a ferv  months  the  following  percentages  of  ammonia  nitrogen  were 
nitrified : 


Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

The  mixtures  f clay 

0 

10 

15 

20 

25 

30 

contained  l sand 

100 

90 

85 

80 

75 

70 

Nitrogen  nitrified 

63 

66 

94 

100 

21 

2.7 

In  the  mixtures  of  high  sand  content  probably  part  of  the  ammonia 
was  lost  by  evaporation.  That  the  low  nitrification  in  the  mixtures 
relatively  rich  in  clay,  on  the  other  hand,  was  solely  due  to  insufficient 
moisture,  was  proved  by  the  following  experiments.  Somewhat  larger 
amounts  of  water  were  added  to  the  mixture  containing  30  per  cent 


1 Com-pt.  rend.  Acad.  Paris,  vol.  109,  1889,  p.  423;  vol.  125,  1S97,  p.  826. 


254 


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clay  and  70  per  cent  sand,  and  now  the  nitrification  proceeded  in  the 
following  manner: 


Per  Cent 

Per  Cent 

Per  Cent 

Per  Cent 

Moisture  content 

10.6 

80 

11.5 

100 

13.2 

100 

14.0 

100 

Nitrogen  nitrified 

Undoubtedly,  the  increase  in  nitrification  which  is  frequently  observed 
as  a result  of  soil  tillage,  is  much  less  due  to  an  increase  in  aeration, 
than  to  an  increase  in  the  water-holding  capacity  and  in  the  moisture 
content  of  the  soil. 

If  the  conditions  are  very  favorable  for  nitrification,  as  is  common 
in  irrigated  soils  in  arid  regions,  this  process  may  assume  unusual 
dimensions.  Strong  evaporation  of  the  soil  moisture  may  cause  ac- 
cumulations of  nitrates  in  the  surface  soil,  so-called  niter  spots,  which 
sooner  or  later  prove  deleterious  to  the  crop.  Most  of  the  nitrate 
formed  itnder  such  conditions  is  derived  from  the  decomposition  of 
humus ; one  half  or  more  of  the  total  nitrogen  content  of  these  soils 
is  sometimes  made  up  of  nitrates.  Heavy  mulching  with  organic  sub- 
stances is  to  be  recommended  for  regulating  the  physical  as  well  as 
the  biological  conditions  of  such  abnormal  soils. 

Assimilation  of  Ammonia  and  Nitrate  Nitrogen. — It  very  rarely 
happens  that  all  the  nitrogen  given  to  the  soil  in  the  form  of  fertilizer 
is  taken  up  by  the  following  crop.  As  a rule,  it  must  be  accepted  as  a 
satisfactory  result  if  approximately  60  to  80  per  cent  of  nitrate 
nitrogen,  50  to  75  per  cent  of  the  nitrogen  of  ammonium  sulfate, 
cyanamid,  and  liquid  manure,  40  to  60  per  cent  of  the  nitrogen  of 
green  manures,  and  20  to  40  per  cent  of  that  in  barnyard  manure  are 
returned  in  the  first  year’s  crop.  Part  of  the  nitrogen  in  the  form  of 
nitrate  is  lost  by  leaching,  but  the  pronounced  inferiority  of  the  organic 
manures  is  mainly  due  to  their  high  content  of  carbonaceous  substances 
which  enable  the  microorganisms  of  the  soil  to  assimilate  the  manure 
nitrogen  as  well  as  that  present  in  mineralized  form  in  the  soil,  and 
in  this  way  to  rebuild  complex  organic  compounds. 

If  large  quantities  of  straw  are  added  to  the  soil,  nitrate  and  am- 
monia are  rapidly  assimilated  by  bacteria  and  fungi.  A new  crop 
planted  soon  after  will  show  all  indications  of  nitrogen  starvation. 
Under  average  conditions  these  processes  are  not  of  very  great  im- 
portance ; about  3 to  5 per  cent  of  nitrate  nitrogen,  and  10  to  25  per 
cent  of  ammonia  nitrogen  may  be  temporarily  side-tracked  in  this 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


255 


manner.  Sooner  or  later  the  assimilated  nitrogen  will  again  be 
mineralized. 

Losses  of  Nitrogen. — The  bad  effects  frequently  caused  by  straw 
and  fresh  stable  manure  have  been  repeatedly  explained  by  the  assump- 
tion that  they  are  due  to  the  detrimental  activities  of  the  denitri- 
fying bacteria.  It  has  been  overlooked  that  an  intensive  deni- 
trification can  take  place  solely  under  strictly  anaerobic  conditions. 
It  is  true  that  the  rapid  formation  of  carbon  dioxide  from  the  straw 
and  the  saturation  of  the  soil  by  heavy  rains  may  temporarily  establish 
anaerobic  conditions  in  an  otherwise  well  aerated  soil.  Under  such 
exceptional  conditions  losses  of  nitrogen  will  occur,  and  occasionally 
this  possibility  may  find  practical  application  as  a means  of  removing 
the  excess  of  nitrate  from  land  showing  niter  spots.  Under  normal 
conditions,  however,  the  denitrification  will  not  cause  serious  losses 
in  the  field.  When  the  soil  becomes  water-logged  as  a result  of  heavy 
rains,  the  losses  by  leaching  are  probably  always  much  larger  than 
those  due  to  denitrification. 

Considerable  quantities  of  ammonia  nitrogen  may  be  lost  by 
evaporation  in  light  calcareous  soils,  but  this  possibility  may  also  be- 
come of  importance  in  soils  of  higher  absorptive  power,  especially  in 
the  application  of  liquid  manure  and  of  cyanamid.  These  substances 
should  at  the  time  of  application  be  thoroughly  mixed  with  or  incor- 
porated in  the  soil. 

Fixation  of  Nitrogen  by  Nodule  Bacteria. — Enough  elementary 
nitrogen  from  the  air  is  continually  assimilated  by  bacterial  action  in 
virgin  as  well  as  in  properly  cultivated  land  that  occasional  losses 
of  nitrogen  are  easily  overcome,  and  the  fertility  of  the  soil  is  main- 
tained or  increased.  The  fixation  of  nitrogen  by  the  bacteria  in  the 
root  nodules  of  leguminous  and  some  other  plants  is  undoubtedly  of 
greatest  importance,  as  was  discussed  on  p.  116.  Figures  58  and  59 
show  a few  characteristic  types  of  root  nodules.  From  the  appear- 
ance of  the  nodules  certain  conclusions  can  be  drawn  concerning  the 
distribution  of  the  bacteria  in  the  soil  and  their  efficiency.  Young 
roots  only  are  invaded  by  the  bacteria,  and  the  more  numerous  and 
efficient  these  organisms  are,  the  more  numerous  and  better  developed 
are  the  nodules  in  the  oldest  parts  of  the  roots.1 

The  bacterial  growth  within  the  nodules  is  visible  in  Fig.  60.  From 
the  vascular  bundle  in  the  center  of  the  root  a sidebranch  is  seen  to 

1 The  normal  root  nodules  are  sometimes  replaced  by  similar  formations  caused  by 
Bad.  tumefaciens,  the  organism  of  crown  gall,  which  is  purely  parasitic.  See  U.  S. 
Dept,  of  Agr.,  Bureau  uf  Plant  Industry  Circular  70. 


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Fig.  58.— Roots  with  nodules  of  (a)  young  alfalfa,  ( b ) young  pea,  (c)  mature  red  clover, 
(d)  mature  soy  bean  (5  nat.  size). 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


257 


enter  the  nodule,  where  it  spreads  around  the  bactei'ial  agglomeration. 
The  carbohydrates  needed  by  the  bacteria  are  brought  to  them  by  this 


a be  d 

Fig.  59. — Roots  with  nodules  of  (a)  red  clover,  (6)  broad  bean,  (c)  lupine,  and  ( d ) alder 

(t  nat.  size). 


a b 

Fig.  60. — Sectional  views  of  a root  nodule,  cut  (a)  lengthwise  and  ( b ) crosswise,  X20 
The  broken  line  indicates  where  the  second  cut  was  made. 

channel,  and  the  nitrogenous  metabolic  products  are  transferred  to  the 
host  plant,  as  far  as  available. 

The  chemistry  of  the  nitrogen  fixation  and  its  products  is  not  yet 


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fully  known;  it  is  probably  all  protein  that  goes  from  the  bacteria 
to  the  host.  The  nodules  themselves  retain  a considerable  part  of 
the  assimilated  nitrogen,  and  this  explains  in  part  the  beneficial  effect 
which  is  often  realized  when  leguminous  plants  are  used  as  forage, 
and  stubble  and  roots  only  are  left  on  the  ground.  If  the  whole  plants 
are  turned  under  as  green  manure  it  may  happen  that  their  carbohy- 
drates will  check  the  mineralization  of  the  nitrogen,  and  the  fertilizing 
effect  will  perhaps  be  lower  than  in  the  other  case.  There  are  also 
economic  reasons  which  make  it  advisable  to  use  the  legumes  as  feed, 
whenever  possible. 

Increase  of  Soil  Nitrogen  by  Leguminous  Crops. — A good  crop  of 
legumes  contains  approximately  100  to  200  lbs.  of  nitrogen  per  acre, 
but  this  amount  does  not  represent  the  quantity  assimilated  from  the 
air.  If  a soil  is  rich  in  nitrate,  much  of  it  is  used  by  the  leguminous 
plants  and  by  the  bacteria,  and  the  nitrogen  fixation  remains  relatively 
low ; but  even  a poor  soil  furnishes  some  nitrate  nitrogen,  although 
the  nitrogen  fixation  is  very  important  in  this  case  and  very  beneficial 
for  the  fertility  of  the  soil.  It  has  been  well  known  for  many  centuries 
that  poor  and  worn-out  soils  can  be  enriched  most  successfully  and  most 
economically  by  an  intelligent  use  of  leguminous  crops,  and  persistent 
use  should  be  made  of  these  very  valuable  abilities  of  the  nodule 
bacteria.  Of  course,  the  assimilated  nitrogen  is  not  clear  gain,  although 
it  is  often  asserted  that  it  may  be  secured  without  cost.  The  actual 
costs  vary  according  to  numerous  calculations  between  5 and  20c.  per 
pound  of  nitrogen,  and  because  the  fertilizing  effect  of  organic  nitrogen 
is  always  below  that  of  ammonium  and  nitrate  nitrogen,  proper  allow- 
ance must  be  made  for  these  differences,  too. 

To  secure  the  highest  possible  benefit  from  a leguminous  crop,  it  is 
necessary  that  the  soil  contains  large  quantities  of  phosphate,  of  potas- 
sium, and  of  lime  if  this  is  needed  by  the  legume  planted.  Only  healthy 
strong  plants  produce  carbohydrates  in  abundance,  and  the  nitrogen 
fixing  bacteria  are  most  active  in  their  root  nodules.  Weak  sickly 
plants  show  very  few  or  no  nodules,  which  fact  demonstrates  clearly 
that  the  relations  between  nodule  bacteria  and  leguminous  plants  are 
truly  symbiotic.  If  the  bacteria  acted  as  parasites,  as  is  sometimes 
asserted,  the  most  numerous  nodules  should  be  expected  on  the  roots  of 
sick  plants,  which  is  contrary  to  the  facts  observed.  Soluble  nitro- 
genous compounds  should  be  absent  in  the  soil,  as  far  as  possible: 
therefore,  it  is  a good  practice  to  use  legumes  as  catch  crops  after 
cereals,  which  have  depleted  the  soil  of  its  nitrate. 

Types  of  Nodule  Bacteria. — If  a legume  is  planted  in  a field  where 
neither  this  nor  related  plant  species  have  grown  before,  it  may  happen 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


259 


that  no  root  nodules  are  formed.  Generally,  nodule  formation  and 
nitrogen  fixation  are  most  satisfactory  if  the  soil  contains,  or  is  sup- 
plied with  the  particular  type  of  bacteria  adapted  to  the  special  kind 
of  legume.  Clover  grows  best  with  clover  bacteria,  peas  with  pea 
bacteria,  etc.  Cross  inoculations  from  one  to  another  legume  are  rare 
with  some,  though  frequent  with  other  types  of  nodule  bacteria.  It 
is  an  open  question  whether  or  not  the  different  types  of  nodule  bac- 
teria should  be  classified  as  separate  species.  The  known  differences 
in  their  general  character  are,  as  a whole,  so  inconspicuous  that  it 
seems  preferable  to  group  the  strains  isolated  from  different  plants 
as  more  or  less  stable  modifications  or  types  of  one  species,  which  was 
described  by  Beijerinck  as  B.  radicicola.  This  holds  true  especially 
for  all  strains  that  cause  root  nodules  on  the  legumes  which  for  cen- 
turies have  been  cultivated  in  Europe.  They  all  have  peritrichous 
flagella,  and  their  cultural  behavior  is  so  much  alike  that  they  can  not 
be  differentiated,  except  by  inoculation  and  agglutination  tests.  Some- 
what greater  differences  become  noticeable  if,  for  instance,  soy  bean 
bacteria  are  compared  with  pea  bac- 
teria, but  here  again  all  cultures 
isolated  from  cultivated  legumes  of 
Asiatic  origin  behave  alike.  They 
have  single,  rarely  several,  polar 
flagella,  and  show  uniform  cultural 
features  which  are  not  very  different 
from  those  of  the  first-named  group.1 
It  may  be  that  these  two  groups 
really  represent  different  species,  but 
so  long  as  their  full  life  history  is  not 
known,  no  final  decision  can  be  made. 

Branched  forms  are  generally  more 
frequent  in  the  peritrichous  group 
(Figs.  2 and  7,  Plate  II),  while  such 
granulated  cells  as  are  shown  in  Fig. 

61  are  common  in  both  groups. 

According  to  their  ability  to  invade  one  or  different  legumes  the 
nodule  bacteria  are  to  be  grouped  as  follows : 

A.  Peritrichous  type,  found  in  cultivated  legumes  of  European  origin: 

1.  Red  clover  (Trifolium  pratense),  Alsike  or  Swedish  clover  (Trifolium  hy- 
bridum),  Crimson  clover  (Trifolium  incarnatum),  White  clover  (Trifolium 
repens),  Cow  clover  (Trifolium  medium),  Berseem  or  Egyptian  clover 
(Trifolium  alexandrinum) . 

1 F.  Lohnis  and  R.  Hansen,  Jour.  Agr.  Research,  vol.  20,  1921,  p.  543;  I.  Shunk, 
Jour.  Bacteriol. , vol.  6,  1921,  p.  239. 


Fig.  61. — Bacteria  from  alfalfa  nodules 
stained,  X1200. 


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2.  Alfalfa  (Medicago  sativa  and  falcata),  Bur  clover  (Medicago  hispida),  Black 

medick  or  yellow  trefoil  (Medicago  lupulina),  White  sweet  clover  (Melilotus 
alba),  Yellow  sweet  clover  (Melilotus  officinalis),  Fenugreek  (Trigonella 
f ornum-gr£ecum) . 

3.  Garden  and  field  pea  (Pisum  sativum),  Hairy  vetch  (Vicia  villosa),  Spring 

vetch  (Vicia  sativa),  Broad  bean  (Vicia  Faba),  Lentil  (Lens  esculenta), 
Sweet  pea  (Lathyrus  odoratus) . 

4.  Garden  bean  (Phaseolus  vulgaris  and  angustifolius),  Scarlet  runner  bean 

(Phaseolus  multiflorus) . 

5.  Lupines  (Lupinus  albus,  angustifolius,  luteus,  and  perennis),  Serradella  (Omi- 

thopus  sativus) . 

B.  Monotrichous  type,  found  in  cultivated  legumes  of  Asiatic  origin: 

1 . Soy  bean  (Soja  max) . 

2.  Cowpea  (Vigna  sinensis),  Peanut  (Arachis  hypogaea),  Japan  clover  (Lespedeza 

striata),  Velvet  bean  (Stizolobium  deeringianum),  Beggarweed  (Des- 
modium  tortuosum),  Kudzu  vine  (Pueraria  thunbergiana),  Lima  bean 
(Phaseolus  lunatus). 

This  grouping  shows  clearly  that  certain  types  of  nodule  bacteria 
are  able  to  produce  root  nodules  on  very  different  plants,  while  other 
strains  display  a remarkably  exclusive  behavior.  Occasionally,  how- 
ever, unusual  cross  inoculations  have  been  recorded,  for  example  be- 
tween Medicago  and  Lupinus,  Phaseolus  vulgaris  and  Vigna  sinensis, 
or  Soja  and  Vigna,  which  are  of  great  scientific  though  of  no  practical 
interest.  The  very  sharp  adaptations  of  the  different  types  of  nodule 
bacteria  to  their  host  plants  are  probably  partly  due  to  their  preference 
for  the  special  degree  of  acidity  that  is  characteristic  of  the  sap  of 
these  plants.  The  minimum  hydrogen-ion  concentration  at  which  the 
bacteria  still  grew  showed,  for  instance,  the  following  interesting  dif- 
ferences in  the  pH  values  d 


Medicago  and  Melilotus  bacteria 

5.0 

Phaseolus  bacteria 

4.3 

Pisum  and  Vicia  bacteria 

4.8 

Soja  bacteria 

3.4 

Trifolium  bacteria 

4.3 

Lupinus  bacteria 

3.2 

Certain  facts  seem  to  indicate  that  in  the  soil  itself  more  or  less 
neutral  strains  of  nodule  bacteria  may  occur,  which  will  gradually 
adapt  themselves  if  a new  kind  of  legume  that  at  first  remains  without 
nodules  is  planted  continuously  for  several  years.  For  all  practical 
purposes,  however,  inoculation  with  adapted  bacteria  is  strongly  to  be 
recommended. 

Asymbiotic  Nitrogen  Fixation. — The  fixation  of  nitrogen  by  Azoto- 
bacter  and  other  soil  organisms,  which  have  been  discussed  in  Chapter 
VII,  4,  can  never  enrich  the  soil  to  such  an  extent  as  is  possible  by 
the  growth  of  leguminous  plants.  Sometimes  a symbiosis  between 
1 E.  B.  Fred  and  A.  Davenport,  Jour.  Agr.  Research,  vol.  14,  1918,  p.  317. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


261 


bacteria  and  algae  may  improve  the  supply  of  carbonaceous  material, 
but  usually  there  is  a decided  lack  of  such  substances  in  the  soil,  and 
the  nitrogen  fixing  bacteria  are  therefore  not  in  a position  to  display 
their  full  abilities.  Approximately  100  parts  of  suitable  carbonaceous 
material  are  necessary  to  furnish  the  energy  for  the  fixation  of  one 
part  of  nitrogen.  Under  average  conditions  3000  to  4000  lbs.  of  organic 
matter  are  annually  available  per  acre.  How  much  of  this  quantity 
is  used  by  the  nitrogen  fixing  bacteria,  is  not  known ; but  it  is  evident 
that  under  unfavorable  conditions  perhaps  10,  and  under  favorable 
conditions  not  more  than  40  lbs.  of  nitrogen  per  acre  and  year  can  be 
expected  from  this  source,  that  is,  as  was  said  before,  1/10  to  1/s  of 
what  may  be  expected  from  the  symbiotic  nitrogen  fixation  in  the 
legumes. 

The  results  of  bacteriological  and  chemical  laboratory  tests,  as  well 
as  of  long  continued  field  experiments  conducted  in  various  parts  of 
Europe,  agree  well  with  these  calculations.  A plus  of  20  to  40  lbs. 
per  acre  and  year  has  been  regularly  observed,  when  all  data  were 
carefully  collected  for  drawing  an  accurate  nitrogen  balance.  Much 
higher  results  have  been  recorded  when  the  nitrogen  content  in  un- 
cropped land  was  determined  after  long  intervals,  as  was  done  in 
Rothamsted  in  1879,  1888,  and  in  1912.  This  soil  showed  an  increase 
in  nitrogen  content  from  0.205  to  0.235,  and  to  0.338  per  cent.1  Un- 
doubtedly, wild  growing  legumes  contribute  to  the  total  effect  in  such 
cases,  and  the  carbon  supply  is  also  much  larger  than  in  cropped  fields. 
In  the  tropics  higher  temperature  is  another  factor  which  may  stimu- 
late the  asymbiotic  nitrogen  fixation  in  the  soil. 

Conditions  of  Nitrogen  Fixation  in  Soil. — Since  the  carbon  meta- 
bolism in  the  soil  affects  the  nitrogen  fixation  probably  more  than  does 
any  other  influence,  it  becomes  again  very  evident  that  the  conservation 
and  regeneration  of  the  humus  content  of  the  soil  requires  most  careful 
consideration.  Many  experiments  have  shown  that  under  favorable 
circumstances,  especially  when  the  temperature  was  not  too  low,  and 
sufficient  time  was  allowed  for  the  various  transformations,  the  applica- 
tion of  sugar  or  of  other  easily  available  carbonaceous  substances 
stimulates  nitrogen  fixation  very  much.  Two  of  such  tests  are  repro- 
duced in  Fig.  62.  Other  promising  experiments  have  been  made  with 
molasses,  with  peat  previously  treated  with  hydrochloric  acid,  and 
with  other  cheap  carbonaceous  materials ; but  no  practical  application 
of  these  results  has  thus  far  become  possible,  and  not  too  much  should 
be  expected  for  the  future. 

1 E.  J.  Russell,  “Soil  Conditions  and  Plant  Growth.” 


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Because  a slightly  alkaline  reaction  is  most  favorable  for  the  growth 
of  the  nitrogen  fixing  soil  bacteria,  regular  liming  of  all  soils  which 
show  a tendency  to  become  acid  is  much  to  be  recommended.  The  ap- 
plication of  basic  slag  or  of  acid  phosphate  also  stimulates  growth  and 
activity  of  Azotobacter  very  distinctly ; but  no  less  important  than 
the  chemical  condition  of  the  soil  is  its  physical  structure.  A soil  in 


Fig.  62. — Buckwheat  and  oats  fertilized  with  sugar,  after  A.  Koch.  Pots  101  and  166 
were  not  treated,  117  and  162  received  sugar. 

perfect  tilth  offers  the  most  suitable  environment  to  the  nitrogen  fixing 
as  well  as  to  all  other  useful  soil  organisms. 

3.  MEANS  OF  REGULATING  THE  ACTIVITY  OF  MICROORGANISMS  IN  THE 

SOIL 

The  bacterial  activities  can  be  regulated  in  the  soil  as  everywhere 
else  by  direct  and  by  indirect  methods.  Direct  methods,  that  is,  soil 
disinfection  and  inoculation,  are  applicable  in  exceptional  cases  only. 
The  normal  way  of  regulating  the  bacterial  activity  in  the  soil  consists 
in  the  intelligent  use  of  all  those  indirect  methods  which  serve  to 
maintain  and  to  increase  the  productivity  of  the  soils.  Tillage,  irriga- 
tion, liming,  manuring,  cropping,  etc.  are  all  of  great  influence  upon 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


263 


the  environmental  conditions  under  which  the  soil  organisms  live  and 
work.  The  better  these  relations  are  understood,  the  better  results 
will  be  secured  in  regulating  the  “life”  of  the  soil,  provided  season 
and  weather  are  favorable  for  the  bacterial  activities  in  the  soil,  as  well 
as  for  the  development  of  the  cultivated  plants. 

Influence  of  Tillage. — Soil  aeration,  moisture  content,  soil  tempera- 
ture, and  the  distribution  of  the  microorganisms  and  their  food  supply 
are  more  or  less  changed  by  every  kind  of  mechanical  soil  treatment. 
Harmful  anaerobic  processes,  such  as  denitrification,  can  not  do  great 
damage  in  a well  tilled,  that  is,  a well  aerated  soil.  On  the  other 
hand,  nitrification,  the  formation  of  carbon  dioxide,  and  other  aerobic 
transformations  are  often  greatly  stimulated  by  plowing  and  cul- 
tivating; but  it  is  less  the  increased  aeration  than  the  change  in  other 
conditions  that  is  of  benefit  to  the  active  organisms.  It  was  pointed 
out  before  that  the  desired  aerobic  processes  are  influenced  very  little 
when  the  oxygen  tension  is  lowered  to  about  one  half  of  normal,  and 
in  regard  to  the  formation  and  transformation  of  humus  such  semi- 
anaerobic  conditions  have  proved  most  favorable. 

The  stimulating  effect  of  plowing  and  cultivating  is  mostly  due  to 
the  increase  in  the  water-holding  capacity  of  the  soil  and  to  the  better 
distribution  of  the  bacteria  and  of  the  organic  residues  in  the  soil. 
If  the  mechanical  treatment  is  as  thorough  as  it  is  in  the  filling  of 
pots  or  in  the  preparation  of  soil  samples  for  laboratoi’y  tests,  a 
marked  increase  in  germ  content  and  in  bacterial  activities  can  always 
be  noted  (see  p.  53).  In  the  field,  excessive  cultivation  or  plowing  at 
the  improper  time  may  also  greatly  stimulate  the  processes  in  the  soil. 
The  rapid  loss  of  humus  as  a result  of  clean  cultivation  is  well  known. 
Nitrogen  losses  will  be  very  large  if  the  natural  tendency  of  nitrifi- 
cation to  reach  a maximum  in  autumn  is  stimulated  by  thorough 
mechanical  treatment  of  the  soil  at  that  time,  and  the  nitrate  formed 
is  then  washed  away  during  a relatively  warm,  rainy  winter. 

If  a soil  is  rich  in  humus  and  full  of  life,  deep  plowing  and  cul- 
tivating is  to  be  recommended.  In  the  opposite  case  the  active  surface 
soil  should  never  be  buried  below  an  inert  subsoil.  Regular  applica- 
tion of  organic  manures,  subsoiling,  and  gradually  deepening  the  fer- 
tile surface  layer  will  greatly  increase  the  productivity  of  such  a soil. 
If  the  soil  is  loose  and  the  climatic  conditions  favor  the  evaporation 
of  water,  this  should  be  checked  as  much  as  possible  by  packing  of 
the  subsurface  and  by  frequent  harrowing,  hoeing,  shallow  cultivation 
or  mulching  of  the  surface  soil. 

Influence  of  Irrigation  and  of  Drainage. — The  normal  bacterial 
processes  go  on  most  satisfactorily  in  a soil  whose  moisture  content 


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is  equal  to  60  to  80  per  cent  of  the  total  water-holding  capacity. 
Naturally,  this  optimum  will  not  persist  regularly,  but  because  of  the 
seasonal  variations  in  bacterial  activities  it  is  very  important  that  in 
spring  and  in  autumn  sufficient,  though  not  too  much,  moisture  should 
be  present  in  the  soil.  If  other  means  of  regulating  the  water  content 
prove  insufficient,  irrigation  and  drainage  must  be  relied  upon,  accord- 
ing to  circumstances.  In  addition  to  changing  the  moisture  of  the 
soil,  both  methods  exert  great  influence  upon  its  temperature,  and  in  a 
lesser  degree  upon  its  aeration.  As  a rule,  a well  drained  soil  is  a 
very  active  soil,  provided  excessive  drainage  has  been  avoided.  A 
temporary  drought  is  not  very  detrimental  to  the  soil  organisms;  they 
become  dormant,  but  are  not  killed.  Frequently  an  increase  in  bac- 
terial activities  occurs  after  the  soil  has  been  thoroughly  dried  and 
remoistened ; in  irrigated  land  this  effect  is  sometimes  very  marked. 
The  improvement  is  partly  due  to  a reduction  in  the  number  of  soil 
protozoa,  which  are  favored  by  excessive  moisture  and  may  do  great 
harm  to  the  bacterial  flora  in  wet  soils.  Better  aeration,  increase  in 
soil  temperature,  changes  in  the  soil  solution  and  in  the  condition  of 
the  soil  colloids  contribute  to  the  favorable  effect. 

Influence  of  the  Application  of  Organic  Manures. — The  regular 
application  of  organic  manures  exerts  in  all  soils  which  are  not  very 
rich  in  humus  the  most  pronounced  influence  upon  the  bacterial  activ- 
ities. Wherever  this  fact  is  neglected,  there  is  an  unavoidable  decline 
in  the  productivity  of  the  land,  as  is  demonstrated  by  the  greatly  de- 
pleted fertility  of  large  areas  in  the  Old  and  in  the  New  World,  which 
were  formerly  highly  productive.  The  most  profitable  use  of  all  organic 
residues,  that  is,  barnyard  manure,  green  manures,  and  crop  residues 
should  be  considered  very  carefully.  The  organic  substances  contained 
therein  represent  food  for  innumerable  soil  organisms,  and  animal 
manure  enriches  the  soil  directly  by  its  high  bactex-ial  content.  Large 
applications  of  straw  may  act  unfavorably  because  of  the  wide  carbon- 
nitrogen  ratio.  As  was  discussed  on  p.  128,  previous  treatment  with 
ammonium  sulfate  or  urine  may  help  to  get  a better  balanced  manure. 
Green  manure  should  not  be  plowed  under  when  the  soil  temperature 
is  high,  since  the  decomposition  would  be  too  rapid  and  great  losses 
would  result.  If  large  quantities  of  it  are  incorporated  into  the  soil 
shortly  before  sowing,  the  seed  may  be  damaged  by  an  abnormal 
growth  of  fungi.1 

Under  natural  conditions  organic  residues  decay  mostly  on  the  sur- 
face, and  it  is  known  that  such  a natural  cover  of  humus  exerts  a very 

*E.  B.  Fred,  Jour.  Agr.  Research,  vol.  5,  1916,  p.  1161. 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS  265 

favorable  influence  upon  physical,  chemical,  and  biological  conditions 
of  the  soil.  Mulching  with  organic  residues  has  given  good  results  in 
orchards  and  on  land  used  in  truck  farming.  Where  the  summer  tem- 
perature is  high  and  the  water  evaporation  strong,  mulching  should  also 
be  tried  in  the  field ; it  keeps  the  soil  moist  and  cool,  and  the  development 
of  the  desired  mellow  structure  is  greatly  facilitated.  In  irrigation 
districts  much  water  can  be  saved  by  this  practice;  even  weeds  may 
serve  a useful  purpose  in  the  form  of  mulch. 

It  has  been  tried  repeatedly  to  prepare  valuable  organic  manures  from 
the  inert  humus  accumulated  in  peat  land.  Chemical  and  bacteriological 
methods  have  been  applied,  but  thus  far  without  conspicuous  success. 
Humogen,  invented  by  W.  B.  Bottomley,  Alphano  humus,  guanol,  and 
other  products  have  been  offered  by  the  trade.  They  have  proved  useful 
in  gardens  and  greenhouses,  but  they  cannot  be  recommended  for  general 
farming  purposes. 

Influence  of  the  Application  of  Mineral  Fertilizers. — The  phosphate 
requirement  of  many  soil  organisms,  especially  of  the  nitrifying  bacteria 
and  of  Azotobacter,  is  distinctly  greater  than  that  of  the  cultivated 
plants.  If  the  soil  is  well  supplied  with  phosphates,  greater  action  may 
be  expected  from  those  organisms,  and  it  seems  possible  that  upon  this 
behavior  tests  may  be  based  which  would  permit  a fairly  accurate  judg- 
ment concerning  the  quantities  of  available  phosphates  present  in  a soil. 
The  reaction  toward  potassium  is  much  less  pronounced,  although  some- 
times quite  noticeable. 

Nitrate  and  ammonium  sulfate  act  unfavorably  upon  nitrogen 
fixation  if  they  are  used  in  very  large  quantities ; but  the  relatively 
small  amounts  generally  applied  are  without  any  effect  or  act  even 
favorably.  Nitrate  and  ammonium  sulfate  influence  the  soil  reaction 
very  much,  especially  if  one  or  the  other  is  used  exclusively  for  a long 
time.  Regular  applications  of  sodium  nitrate  tend  to  increase  the 
alkalinity  of  the  soil,  while  ammonium  sulfate  makes  the  soil  acid  and 
causes  fungi  to  grow  in  abundance. 

Effect  of  Lime  and  of  Sulfur. — An  almost  neutral  soil  reaction,  not 
far  from  pH  = 7,  is  generally  best  for  cultivated  plants  and  for  soil 
organisms ; but  exceptions  do  occur.  Certain  cultivated  plants 
prefer  a distinctly  alkaline  soil,  while  others  display  the  opposite  be- 
havior. Too  much  alkali  creates  unfavorable  chemical  and  physical 
conditions  in  the  soil,  which  can  be  improved  by  the  use  of  acids  or 
acid  salts,  as  well  as  by  strengthening  the  bacterial  acid  formation  by 
the  application  of  organic  manures  or  of  sulfur. 

The  presence  of  Azotobacter  and  its  tendency  to  grow  in  laboratory 
tests  weakly  or  vigorously  has  been  recommended  as  a basis  for  classi- 


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fying  soils  according  to  their  lime  requirement  and  general  produc- 
tivity. The  results  obtained  have  been  fairly  satisfactory,  but  it  seems 
as  if  the  simpler  determination  of  the  hydrogen-ion  concentration  fur- 
nishes approximately  the  same  information.  Undoubtedly,  the  careful 
control  and  the  adjustment  of  the  soil  reaction  will  gain  in  importance, 
after  the  influence  of  this  factor  upon  plant  growth  and  bacterial 
activity  has  been  investigated  more  thoroughly. 

Excessive  liming  should  be  avoided,  because  the  intensive  stimula- 
tion of  the  biochemical  processes  may  deplete  the  fertility  of  the  soil 
very  seriously.  Temporarily  the  germ  content  of  a field  that  was 
heavily  supplied  with  slaked  lime  may  show  a decrease,  due  to  the 
caustic  action  of  the  lime,  but  an  increase  in  number  and  activity  will 
ahvays  follow. 

Influence  of  Cropping. — Undoubtedly  the  microflora  of  the  soil  is 
greatly  influenced  by  the  crops  growing  on  the  land,  but  not  much 
is  known  at  present  about  these  correlations.  Legumes  have  generally 
been  found  to  act  most  favorably,  and,  as  a rule,  the  cultivated  crops 
(potatoes,  corn,  beets)  stand  next  to  them.  Other  plants,  for  in- 
stance mustard,  seem  to  display  a distinctly  detrimental  influence  upon 
nitrification  and  other  desirable  bacterial  activities  in  the  soil.  The 
growing  plants  act  upon  the  microflora  of  the  soil  directly  and  in- 
directly. Their  roots  are  always  covered  by  associations  of  micro- 
organisms, and  the  whole  area  occupied  by  the  root  system  is  supplied 
with  organic  substances  originating  in  living  and  in  dying  cells,  which 
favor  the  growth  of  specific  groups  of  bacteria.  Furthermore,  the 
root  activity  changes  more  or  less  the  soil  reaction,  the  concentration 
of  the  soil  solution,  the  solubility  of  inorganic  and  of  organic  soil 
constituents,  the  physical  structure,  and  the  water  content  of  the  soil. 
All  these  influences  together  must  necessarily  modify  the  bacterial 
activities  in  the  soil. 

The  physical,  chemical,  and  biological  conditions  of  a soil  are  most 
likely  to  be  harmed  by  the  continuous  growing  of  one  or  a few  crops 
on  the  same  land.  For  maintaining  the  fertility  of  the  soil  it  is  much 
better  if  mixed  crops  are  grown,  as  on  meadows  and  pastures,  or  a 
proper  crop  rotation  is  followed.  If  soil  is  carefully  tested  after  such 
different  crops  as  legumes,  potatoes,  or  wheat  have  grown  upon  it, 
it  is  usually  not  very  difficult  to  find  out  that  its  structure  as  well  as 
its  bacteriological  qualities  are  best  in  the  first  and  worst  in  the  last 
case.  It  is  a good  practice,  now  widely  adopted  in  Europe,  to  grow 
cereals  not  oftener  than  every  second  year  on  the  same  field  in  regular 
rotation  with  legumes,  potatoes,  sugar  beets,  hemp,  and  other  non- 
cereals. Economic  and  climatic  conditions  do  not  permit  the  general 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


267 


adoption  of  such  a practice  in  America,  but  it  is  fortunate  that  corn 
evidently  does  not  act  as  unfavorably  upon  the  soil  and  its  flora  as 
do  wheat,  oats,  and  barley,  provided  it  is  not  planted  too  frequently 
on  the  same  field.  Furthermore,  in  American  farming  more  leguminous 
plants  can  be  used  as  catch  crops  and  with  better  results  than  in 
Europe,  thus  alleviating  the  disadvantageous  effects  exerted  by  a long 
succession  of  cereal  crops. 

Influence  of  Fallowing. — If  the  climate  is  not  too  extreme  and 
crops  are  grown  in  proper  rotation,  it  is  generally  possible  to  produce 
a profitable  crop  every  year  without  leaving  the  field  from  time  to 
time  in  fallow  for  a full  year ; but  if  the  soil  is  very  raw  and  the 
climatic  conditions  so  unfavorable  as  not  to  permit  a careful  working 
of  the  ground  in  spring  or  in  fall,  an  occasional  fallowing  will  greatly 
improve  the  physical  and  bacteriological  conditions  of  the  soil.  In 
semi-arid  regions  lack  of  water  may  make  it  impossible  to  raise  a 
profitable  crop  every  year,  and  here  the  fallow  may  help  to  secure 
a better  structure  of  the  soil  and  to  store  so  much  water  in  it  that  the 
soil  organisms  as  well  as  the  second  year’s  crop  are  better  supplied. 
In  earlier  times  the  fallow  was  considered  to  be  the  only  reliable  means 
of  getting  soil  under  humid  conditions  in  good  tilth ; but  at  present 
it  is  well  known  that  this  result  can  be  reached,  as  a rule,  by  less 
expensive  methods. 

Soil  Disinfection. — Another  exceptional  means  of  regulating  the 
soil  flora  is  the  so-called  soil  disinfection,  which,  contrary  to  the  treat- 
ments thus  far  discussed,  is  destined  to  act  directly  upon  the  organisms 
in  the  soil.  It  needs  hardly  to  be  emphasized  that  a real  soil  disin- 
fection is  neither  possible  nor  desirable.  Merely  a partial  disinfection, 
a thorough  change  in  the  microflora  of  the  soil  is  intended  and  can  be 
realized.  Numerous  physical  and  chemical  methods  have  been  tried 
or  are  in  use  for  these  purposes. 

The  burning  of  field  and  forest  soils,  as  well  as  of  peat  land,  prac- 
ticed since  ancient  times,  and  the  more  modern  treatment  of  green- 
house soil  by  steam  or  hot  water  represent  the  physical  methods 
available;  their  biological  effects  are  supplemented  or  even  surpassed 
by  the  resulting  changes  in  the  physical  structure  of  the  burned  or 
steamed  soil. 

Many  chemical  substances  have  been  tried  for  soil  disinfection 
in  greenhouses  and  in  the  field,  but  only  a few  are  of  practical  value. 
These  are  carbon  bisulfide,  formaldehyde,  and  toluol.  Figure  63  shoAvs 
how  the  growth  of  grapevines  was  improved  by  such  a treatment  in 
a soil  which  had  become  “sick”  because  of  long  continued  use  for 
growing  grapes.  Other  substances  which  have  been  tried  more  or 


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less  successfully  are  chloride  of  lime,  arsenic  and  arsenate,  sulfates 
and  other  salts  of  iron,  copper,  manganese,  and  zinc,  ether,  benzene, 


Fig.  63. — Effect  of  soil  disinfection,  after  Russell  and  Petherbridge  (a)  sick  soil,  ( b ) same 
soil  steamed,  (c)  treated  with  formaldehyde. 

xylol,  and  different  phenol  preparations.  Slaked  lime  applied  in  large 
quantities  exerts  a similar  effect. 

A great  reduction  in  the  number  of  organisms  living  in  the  soil 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


269 


is  the  first  marked  result  from  all  these  treatments.  As  far  as  these 
organisms  are  pathogenic  to  plants  or  detrimental  to  the  useful  micro- 
flora of  the  soil,  their  destruction  is  of  great  benefit.  The  normal  soil 
bacteria  always  show  recovery  after  a few  weeks,  and  because  they 
are  freed  from  such  enemies  as  the  soil  protozoa,  they  multiply  very 
rapidly  making  use  of  the  substances  present  in  the  killed  organisms 
and  liberated  by  the  heat  or  by  chemical  action  from  the  inert  material 
in  the  soil.  Especially  the  solubility  of  the  soil  nitrogen  is,  as  a rule, 
greatly  increased.  Occasionally  unfavorable  results  are  observed  which 


Fig.  64. — Cultures  for  legume  inoculation  (f  nat.  size). 


are  caused  by  acids  or  other  compounds  freed  in  the  accelerated  decom- 
position of  humus.  Liming  checks  the  first  effect,  but  not  the  second 
one. 

It  is  known  that  minute  quantities  of  otherwise  poisonous  sub- 
stances may  act  as  stimulants  upon  higher  and  lower  organisms,  there- 
fore, the  last  remnants  of  the  substances  used  for  soil  disinfection 
may  act  in  this  manner;  but  this  stimulating  effect  can  not  be  of  very 
great  importance,  since  steaming  improves  the  productivity  of  many 
soils  almost  in  the  same  manner  as  does  a treatment  with  formaldehyde 
or  other  chemical  substances. 


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Soil  Inoculation. — For  enriching  the  soil  with  useful  organisms 
barnyard  manure  and  compost  have  been  used  since  ancient  times,  but 
not  before  Pasteur  had  directed  the  general  attention  to  these  biological 
problems,  has  it  been  known  that  the  beneficial  effect  of  such  a treat- 
ment is  at  least  partly  due  to  the  high  germ  content  of  these  manures. 
It  is  not  to  be  denied  that  some  experiments  have  given  and  will  give 
results  which  apparently  do  not  support  this  statement ; but  a marked 


Fig.  65. — Serradella  (Ornithopus  sativus)  (a)  not  inoculated,  ( b ) inoculated  (1  nat.  size) 


improvement  of  the  biological  soil  conditions  has  frequently  been 
recorded,  confirming  the  practical  experiences  that  stable  manure  and 
compost  are  the  best  means  of  getting  ‘'life”  into  the  soil. 

Pure  cultures  or  mixed  cultures  of  certain  soil  organisms,  especially 
of  nitrogen  fixing  bacteria,  have  been  tried  for  soil  inoculation.  A 
few  promising  and  many  unsatisfactory  results  have  been  obtained. 
If  it  is  kept  in  mind  that  the  bacteria  or  their  spores  or  gonidia  are 
present  nearly  everywhere,  but  that  a good  development  can  not  take 
place  except  under  favorable  conditions,  it  is  almost  self-evident  why 


BACTERIA  AND  RELATED  MICROORGANISMS  IN  SOILS 


271 


soil  inoculation  rarely  succeeds.  If  the  environmental  conditions  are 
favorable,  the  useful  bacteria  will  be  present  in  the  soil ; if  they  are 
missing,  it  is  almost  invariably  due  to  unfavorable  conditions,  which 
would  first  have  to  be  improved.  Alinite,  All  Crops  Soil  Inoculum, 
Bacto  Natural,  U-cultures,  Phosphogerm  are  the  names  of  some  com- 
mercial preparations  which  have  been  or  are  widely  advertised  for 
soil  inoculation,  usually  under  very  exaggerated  claims.  Bac-Sul  is 
sulfur  inoculated  with  sulfur  oxidizing  bacteria,  which  is  said  to  be 
much  more  rapidly  oxidized  in  the  soil,  because  here  the  specific  organ- 
isms are  usually  present  in  small  numbers  only. 


Fig.  66. — Soy  beans  (Soja  max.)  inoculated  (left),  and  not  inoculated  (right). 


Plant  Inoculation.— If  pure  cultures  of  nodule  bacteria  are  used  for 
inoculating  leguminous  plants,  good  results  can  be  expected,  because 
the  growing  plant  offers  the  most  suitable  environmental  conditions. 
After  the  same  kind  of  legume,  or  one  which  permits  cross-inoculation, 
has  grown  repeatedly  on  the  same  field,  normal  nodules  are  produced 
in  abundance,  because  the  soil  is  gradually  enriched  with  the  special 
type  of  nodule  bacteria;  but  if  there  is  any  doubt  as  to  the  presence 
of  enough  bacteria  of  the  desired  type,  the  seed  should  be  inoculated 
to  assure  prompt  nodule  formation  and  vigorous  nitrogen  fixation. 

Before  pure  cultures  became  available  for  this  purpose,  soil  was 
frequently  taken  as  inoculum  from  fields  where  a good  crop  of  the 
special  kind  of  legume  had  been  grown.  This  practice  is  still  to  be 
recommended  when  no  reliable  pure  culture  can  be  secured,  provided 


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the  soil  does  not  contain  too  many  weeds  and  plant  diseases.  As  a rule, 
however,  efficient  pure  cultures  for  legume  inoculation  can  easily  be 
secured  at  present  from  Agricultural  Experiment  Stations,  Agricul- 
tural Departments,  and  from  many  commercial  firms.  A number  of 
them  are  shown  in  Fig.  64.  Many  different  trade  names  have  been 
invented,  such  as  Azotogen,  Farmogerm,  Nitragin,  Nitrobacterine, 
Nitroculture,  Westrobac,  etc.  The  cultures  are  furnished  in  liquid 
form,  on  agar,  or  in  soil.  Since  good  soil  is  undoubtedly  the  most 
suitable  of  the  three  substrates,  such  cultures  give  generally  the  best 
results.  If  the  preparations  are  only  a few  days  or  weeks  old,  their 
quality  depends  exclusively  on  the  care  with  which  the  cultures  have 
been  selected  and  propagated. 

Successful  inoculation  increases  the  crop  considerably,  as  may 
be  seen  from  Figs.  65  and  66 ; the  cost  is  very  moderate.  Growing  of 
inoculated  legumes  is  one  of  the  best  means  of  maintaining  and  increas- 
ing the  productivity  of  the  soil. 


INDEX 

Illustrations  are  indicated  by  a * before  page  number 


A 

Abbe’s  condenser,  76 
Accumulation  experiments,  69 
Acetic  acid,  119,  127,  154,  179,  202 
Acid  formation,  119,  *153,  176,  201,  226 
Acidity,  44,  128,  175,  201,  249 
Acids,  effect  of,  83,  133,  239,  253 
transformation  of,  126,  202 
Actinomycetes,  38,  113,  191,  223,  239,  248 
Actinomyces  chromogenes,  *89 
Activated  sludge.  216,  219 
Adaptation,  39,  S5 
Aeration  of  soil,  238,  245,  263 
Aerobic,  *48 
Agar,  69 

Agglutination,  142,  259 
Aggressin,  141,  142 
Air,  filtration  of,  82 
germ  content  of,  62,  166,  189,  198 
pressure,  49 
Alanin,  99,  207 

Alcohol  formation,  122,  155,  180,  206 
test,  175 

Alder  nodules,  113,  *257 
Alexin,  142 
Alfalfa  nodules,  *256 
silage,  153 

Algae,  52,  116,  238,  239,  261 
Alinite,  271 

All  crops  inoculum,  271 
Alphano  humus,  265 
Amino  acids,  assimilation  of,  105,  231 
transformation  of,  99,  129,  203,  205,  231 
Ammonia,  assimilation  of,  *43,  105,  231, 
254 

evaporation  of,  232,  255 
formation  of,  *101,  181,  203,  205,  230, 
251 

nitrification  of,  102-104,  253 
oxidation  of,  97 


Ammonium  sulfide,  134 
Amoebae,  38,  *242 
Anabaena,  113 

Anaerobic  bacilli,  48,  124,  204,  217,  223 
cultures,  *72 
Anaerobiosis,  *48 
Anaphylaxis,  144 
Anilin  dyes,  76 
Animalcula,  5 
Antagonism,  55 
Anthrax,  *22,  144 
Antibodies,  141 
Antiformin,  84 
Antisepsis,  77,  82-85 
Antitoxins,  141 
Ardisia,  113 
Arnold  sterilizer,  *73 
Aroma  production,  192,  206 
Arsenate,  268 
Arsenic,  268 
Arthrospores,  31 
Asepsis,  79 
Asparagin,  *43,  99 
Aspergillus,  *31,  37 
Autoclave,  *73 
Autolysis,  *32 
Autotrophic,  40 
Azotobacter,  *15,  37,  *115,  233 
nitrogen  content  of,  40 
Azotofication,  97 
Azotogen,  272 

B 

Bacillaceae,  37 
Bacillus,  36 
B.  abortus,  162 

acidi  lactici,  119,  127 
acidi  urici,  100 
acidophilus,  187 

aerogenes,  121,  127,  150,  160,  191 


273 


274 


INDEX 


B.  amylobacter,  *30,  115,  *123,  124,  191, 
233 

anthraeis,  *22,  144 
botulinus,  139,  157,  208 
casei,  *19,  121 
casei  limburgensis,  205 
coli,  58,  *74,  100,  121,  127,  150,  i60,  191 
cyanogenes,  *89 
denitrificans,  108 
erythrogenes,  *88,  100,  127 
fluorescens,  58,  *89,  99,  100,  108,  127, 
150,  160,  191,  223 
herbicola,  150 
lactis,  *120 
lactis  acidi,  120,  121 
lactis  viscosum,  116,  122,  165,  233 
malabarensis,  *19 

mesentericus,  59,  88,  99,  122,  127,  151, 
191 

methanicus,  130 

mycoides,  54 

oligocarbophilus,  130 

Pasteuri,  100,  223 

petasites,  116 

pneumoniae,  121 

prodigiosum,  *74,  89,  100,  127 

proteus,  *18,  54,  98,  100,  127,  160,  223 

putidum,  108 

putrificus,  *30,  98,  124,  223 
pyocyaneum,  89,  163 
radicicola,  111,  259,  *260 
radiobacter,  108,  112,  116 
subtilis,  *30,  89,  99,  127,  151,  191 
Stutzeri,  108 
syncyaneum,  *89 
tumefaciens,  255 
typhosus,  121,  150 
vernicosum,  46 
vulgare,  127 
Bacteriaceae,  37 
Bacteria  filters,  *81 
Bacterial  toxins,  102,  139,  140,  208 
Bactericidal  action  of  blood,  141 
of  milk,  167 
Bacteriolysis,  142 
Bacteriophagy,  56 
Bacterium,  16,  36 
Bacto  Natural,  271 
Barnyard  manure  (see  Manure) . 

Bean  nodules,  *257 
Beet  roots,  150,  158 


Beet  tops,  156 
Beggiatoa,  136 
Benzene,  268 
Benzoic  acid,  85,  100,  234 
Berkefeld  filter,  *81 
Biological  milk  tests,  *172-*176 
soil  tests,  243 
Bitter  taste,  207 
Blind  cheeses,  207 
Blood,  bactericidal  action  of,  141 
dried,  decomposition  of,  252 
tests,  143 

Blown  cheese,  *208 

Blue  Dorset  cheese,  204 

Bone  meal,  252 

Botulism,  139 

Branching,  *20 

Bread,  slimy,  122 

Brie  cheese,  197,  205,  206,  214 

Broth,  73 

Brown  hay,  152 

Brownian  movement,  23 

Budding,  *20 

Burning  effect  of  manure,  225,  253 
of  soils,  267 

Butter,  germ  content  of,  *17,  188-196 
quality  of,  191-194 
paper,  189 

Butyric  acid,  123,  154,  193,  206,  226 
bacteria,  *8,  123,  191,  223 

C 

Cabbage,  germ  content  of,  63,  150,  157 

Cadaverin,  205 

Calcium,  42 

Calcium  cyanamid,  101 

Calves’  feces,  germ  content  of,  186 

Camembert  cheese,  197,  203,  205,  214 

Canada  balsam,  76 

Caprinic  acid,  207 

Caproic  acid,  206,  207 

Caprylic  acid,  207 

Capsule,  *22 

Carbohydrates,  assimilation  of,  131 
fermentation  of,  118-123,  225 
Carbolic  acid,  84 

Carbonates,  transformation  of,  132,  227 
Carbon  bisulfide,  267 
Carbon  dioxide,  action  in  soil,  245,  246 
antiseptic  effect  of,  83 
assimilation,  129,  136 


INDEX 


275 


Carbon  dioxide,  formation  of,  50,  129, 
157,  180,  207,  227,  245 
Carbon  metabolism,  *117,  245 
monoxide,  129 
nitrogen  ratio,  94,  12c 
sources,  41 

Carriers  of  diseases,  140,  163,  198 
Casein  decomposition,  181,  201-204,  207 
Catalase  test,  *174,  196 
Catch  crops,  267 
Cattle  manure,  230 
Ceanothus,  113 
Cell  composition,  40 
division,  25 
inclusions,  21 
morphology,  *15-24 
structure,  *21-24 
wall,  22 

Cellulose,  decomposition  of,  *125,  *225- 
227,  *246-248 
digestion  of,  159 
Cement  decomposition,  433 
Centrifugation,  170 
Cephalotrichic,  *24 
Certified  milk,  169 
Cesspool,  216 
Chain  formation,  *20 
Chalk  agar,  *176 
Chamberland  filter,  *81 
Cheddar  cheese,  197,  *199,  203,  204,  208, 
213 

Cheese,  acidity  of,  201,  211 
curing  of,  199,  211 
flavor  of,  206 
gassiness  of,  *208 
germ  content  of,  *17,  65,  197-214 
inoculation  of,  212-214 
pigmentation  of,  209 
poisoning,  208 
water  content  of,  197 
Cheesy  flavor,  192,  207 
Chemical  treatment  of  cream,  195 
of  manure,  235 
of  milk,  184 
of  soil,  267 
of  water,  220 
Chemotherapy,  145 
Chitin,  102 
Chlamydospores,  32 
Chloride  of  lime,  84,  268 
Chlorophyceae,  116 


Chromatin,  40 
Chromidia,  22 
Cieddu,  186 
Cilia,  *23 
Ciliates,  38 
Citrates,  127 

Cladosporium  butyri,  193 
Clarification  of  milk,  136 
Classification,  34-38 
Clostridium,  *30 
butyricum,  *123 
Pastorianum,  115 
Clover  nodules,  *256,  *267 
silage,  153 
Coal,  *117 
Coccaceae,  37 
Cocci,  *16 

Cold  curing,  199,  211 
Cold,  effect  of,  51,  168 
Cold  storage,  190,  193 
Colonies,  *26-28 
Colostrum,  208 
Comma  bacilli,  *15 
Comptonia,  113 
Complement,  143 
Compost,  270 
Concentrated  feed,  150 
Concentration,  46 
Concrete  decomposition,  133 
Condensed  milk,  175 
Congo  red,  88 
Conifers,  113 
Conidia,  *31 
Conjugation,  29 
Conjunction,  *29 
Contact  beds,  219 
Contact  preparates,  27,  *19S 
Contagium  animatum,  6 
Copper  sulfate,  84,  268 
Copulation,  29 
Coriaria,  113 

Com,  germ  content  of,  63,  150 
silage,  153 
Cotton  wool,  82 
Coulommiers  cheese,  214 
Counting  methods,  67,  68,  172,  239,  240 
Cowdung,  germ  content  of,  *222 
Cream,  germ  content  of,  189 
pasteurization,  189,  195 
ripening,  *196 
Crenothrix,  138 


INDEX 


276 

Crop  residues,  245,  263 
Crops,  influence  of,  266 
Crown  gall,  255 
Cryophilic,  51 
Cucumbers,  157 
Curd,  germ  content  of,  198 
test,  *175,  208,  210 
treatment  of,  211 
Curdling  of  milk,  177,  *180 
Cyanamid,  101,  252 
Cvanophyceae,  113,  116 
Cycadeae,  113 
Cycle  of  carbon,  *117 
of  matter,  *2 
of  nitrogen,  *95 
of  sulfur,  *133 
Cysts,  31-33 
Cytology,  21 

D 

Dadhi,  186 
Dairy  bacteriology,  9 
Dairy  utensils,  164,  165,  181-183,  189,  212 
Decay,  94 
Degeneration,  19 
Dematium,  37 

Denitrification,  97,  107,  *108,  233,  255,  263 
Digestion,  159 

Digestive  tract,  bacteria  in,  63,  159,  186 

Dilution  method,  68 

Diphtheria,  144,  171 

Dirt  in  milk,  170 

Diseases,  bacterial,  1,  138 

Disinfection,  78,  216,  220,  224,  *267 

Distilled  water,  43 

Drainage,  263 

Drought,  47,  264 

Dry  cultures,  196,  214,  272 

Drying  effect,  47,  80,  264 

Dry  rot  of  potatoes,  *158 

Dysentery,  216 

E 

Ecto-enzymes,  99 
Ecto-toxins,  140 
Edam  cheese,  203,  204 
Efficiency  of  microorganisms,  *17,  86,  240 
Elaeagnus,  113 
Elasticotropism,  54 
Elective  cultures,  69 
Electric  treatment  of  milk,  85 
of  silage,  156 


Electricity,  53 

Emmeiital  cheese,  197,  199,  202-204,  213 
Endo-enzymes,  99 
Endo-spores,  *29,  *30,  58 
Endo-toxins,  140 
Environment,  4,  39,  87 
Enzymes,  43,  94,  99,  159 
in  cheese,  203 
in  silage,  155 
Ericaceae,  113 
Ether,  268 

Evaporated  milk,  175 
Eyes  in  cheese,  207 

Excrements,  decomposition  of,  229,  230 
germ  content  of,  63,  160,  *222 

F 

Fairy  rings,  28 
Fall  maxima,  238,  *240 
Fallow,  238,  243,  267 
Farmogerm,  272 
Fat,  123 

decomposition  of,  123,  181,  193,  194,  205 
formation  of,  206 
oxidation  of,  194 

Fecal  contamination,  160,  163,  170,  182, 
200,  208,  216 

Feces,  decomposition  of,  229,  230 
germ  content  of,  63,  160,  *222 
Feeding  and  milk  contamination,  166 
Feldspar,  132 
Fermentation,  8,  94 
tests,  *175,  208 

Fermented  milks,  123,  *185-*1S7 

Fertility  of  soils,  114,  247,  258,  263 

Field  experiments,  77,  243 

Fiili,  122,  187 

Filterable  vira,  82 

Filtration,  *80-82 

Fish  guano,  131 

Fishy  flavor,  192 

Fission,  25 

Flagellates,  38 

Flagellation,  *23 

Flax,  124,  150 

Flies,  166,  219 

Formaldehyde,  185,  235,  267 
Formic  acid,  127,  179,  202 
Food  and  mouth  disease,  82,  171 
Foodstuffs,  149-160 
Food  supply,  42 


INDEX 


Freezing,  effect  of,  51,  238 
Fuchsin,  76 

Fungi  imperfecti,  37  (see  Molds) 

G 

Gallionella,  138 

Gas  formation  in  cheese,  207- *209 
in  hay,  93 
in  manure,  227 
in  milk,  180 
in  sauerkraut,  157 
in  soil,  130,  245,  250 
Gelatin,  69 
Gemmae,  32 
Geotropism,  53 
Germ  content  of  air,  62,  166 
of  butter,  *17,  188-196 
of  cheese,  *17,  *197-214 
of  feces,  63-160,  *222 
of  foodstuffs,  149-160 
of  manure,  65,  *221-236 
of  milk,  *17,  26,  *64,  161-176 
of  plants,  62,  149 
of  rennet,  198,  200 
of  soil,  *60,  237-272 
of  water,  62,  165,  189,  215,  220 
Germination,  *30 
Gervais  cheese,  *199,  205,  206 
Giant  colonies,  *27 
Gioddu,  186 
Glycerol,  123,  193,  206 
Glycin,  100,  207 
Glycogen,  41 
Gonidangia,  *31 
Gonidia,  31 

Gorgonzola  cheese,  204 
Grain,  germ  content  of,  150 
Grana  cheese,  214 

Granulobacillus  saccharobutyricus,  35 

Granulobacter  pectinovorum,  124 

Granulose,  41 

Grass  bacilli,  150,  191 

Green  manuring,  109,  245,  246,  251 

Green  sand,  133 

Grusavina,  186 

Guaiacol,  184 

Guanol,  265 

Gypsum,  137,  235 

H 

Hanging  agar  block,  75 
Hanging  drop,  *75 


Hard  cheeses,  197 
Harz  cheese,  199 
Hay  bacilli,  *30,  151 
germ  content  of,  150-152 
ignition  of,  92,  152 
making,  151 

Heat  generation,  51,  91,  152,  226,  228 

Heat  resistance,  59,  60,  79 

Heliozoa,  38 

Hemolysis,  143 

Hemp,  124,  150 

Heterotrophic,  40 

Hexa-meth /lene-tetramin,  235 

Hippophae,  113 

Hippuric  acid,  100 

History  of  bacteriology,  5-11 

Horn  meal,  252 

Horse  manure,  228,  230 

Hot  air  sterilization,  *74,  80,  165 

Humogen,  265 

Humus,  composition  of,  118,  127 
conservation  of,  261,  264 
decomposition  of,  129,  248 
formation  of,  127,  228,  247 
Huslanka,  186 
Hydrogen,  130,  180,  227 
bacillus,  125 
peroxide,  84 
sulfide,  134,  205 

I 

Ignition,  spontaneous,  92,  152 
Immersion  lens,  76 
Immunity,  141 
Incubation,  140 

India  ink  preparations,  *70,  *76 
Indol,  205 
Infection,  140 

Inflammation  of  the  udder,  162,  171 
Infusoria,  7,  38 
Inoculation  of  cheese,  212-214 
of  legumes,  *271-272 
of  manure,  235 
of  sauerkraut,  157 
of  silage,  156 
of  soil,  *270 

Inorganic  respiration,  47,  50 
Insects  in  soil,  248 
Intestinal  flora,  179,  160,  186 
lactic  acid  bacteria,  119,  200 
Involution,  19 


278 


INDEX 


Iron  bacteria,  *137 

Iron  in  dairy  products,  182,  189,  193,  209 
metabolism,  137 
sulfate,  268 
Irrigation,  218,  263 
Isolation,  69 

K 

Kefir,  123,  *187 
Kieselguhr  filter,  *81 
Koumiss,  123,  187 

L 

Lactic  acid  bacteria,  *8,  *119-121,  *178 
formation,  119,  153,  177,  202 
micrococci,  120,  178,  191,  200,  204 
oxidation,  127,  202 
streptococci,  *120 
in  butter,  189,  190 
in  cheese,  200,  204 
in  intestines,  160 
in  milk,  176-180 
on  plants,  150 
in  silage,  155 
Lactobacilli,  *120 
in  butter,  191 
in  cheese,  200,  204 
in  intestines,  160,  187 
in  milk,  178-180,  186 
on  plants,  150 
in  silage,  154 
Lactobacillus  casei,  121 
pentoaceticus,  155 
Leaves,  decomposition  of,  246 
nodules  of,  113 
Lebben,  186 
Lecithin,  131 

Legumes,  crop  rotation,  266 
inoculation,  *270-272 
nitrogen  fixation,  109,  258 
root  nodules,  *256-260 
Lepargyrea,  113 
Leptothrix,  138 
Leucin,  99 

Leucocytes,  141,  167,  *171,  *173 
Leuconostoc,  *22,  122 
Life  cycles,  34 
Light,  influence  of,  52 
production  of,  *90 
Limburger  cheese,  203,  205 
Lime,  83 


Lime,  milk  of,  83,  220 
Liming  effect,  242,  262,  265,  268 
Liquid  manure,  conservation  of,  234,  235 
effect  of,  231 
Literature,  11 

Litter,  decomposition  of,  229 
germ  content  of,  166,  222,  232 
Little  plate  method,  172 
Local  varieties,  65,  206 
Locomotion,  23 
Long  whey,  122 
Lophotrichic,  *24 
Lupine  nodules,  *257 

M 

Macrocyst,  33 
Malates,  127 
Malta  fever,  162 
Manganese,  137,  268 
Mannitol,  122,  155 
Manure,  application  of,  245 
artificial  preparation  of,  128 
conservation  of,  234^236 
decomposition,  131,  224r-236,  249,  251 
effect  of,  131,  224,  245,  254,  270 
germ  content  of,  65,  *221-236 
heat  production  in,  228 
nitrification  in,  104 
rotting  of,  106,  224-236 
Margarine,  191 
Mastigophora,  38 
Mastitis,  162,  *171 
streptococci,  162,  *171 
Matzoon,  186 
Measuring  bacteria,  16 
Meat  substrates,  73 
Mechanical  treatment,  53 
Media,  73,  239,  243 
Membrane,  22 
Mercury  chloride,  84 
Metatrophic,  40 
Methane,  130,  227 
bacillus,  124 

Methods,  67-76,  239,  243 
Methylene  blue,  76 
reduction  test,  *172-174,  185 
Mezzoradu,  186 
Mica,  132 
Microaerophilic,  48 
Micrococcus,  35 

casei  liquefacieas,  2C5 


INDEX 


279 


Micrococcus  lactis  acidi,  119 
pituitoparus,  122 
pyogenes,  121,  162 
Microcysts,  *31 
Micron,  16 

Microscopic  counts,  67,  172 
tests,  *75,  *171,  184 
Milk,  bactericidal  action  of,  8,  176-183 
clarification  of,  166 
curdling  of,  177,  180 
discoloration  of,  *89,  *182 
enzymes  of,  184 
fat,  decomposition  of,  181 
fermentation  tests,  *175,  208 
fermented,  123,  *185-*187 
filtration  of,  82,  167 
germ  content  of,  *17,  26,  *64,  161-176 
pasteurization,  79,  85,  170,  184,  212 
powder,  196 
ripening,  211 
sterilization,  80,  181 
testing,  171-176 
viscosity,  183 

Milking,  effect  on  germ  content,  163 
Milking  machines,  164 
Mineralization  of  bacterial  cells.  108 
of  organic  residues,  130,  245,  246,  250- 
254 

Mineral  requirements,  42 
Mineral  substances,  transformation  of, 
130-138,  245 
Mixed  growth,  53,  205 
Moisture  (see  Water  content). 

Molasses,  236 
Molds,  37,  50 
in  air,  189 

in  dairy  products,  191,  200,  206 
in  feed,  149 
in  manure,  45,  223 
in  soil,  238,  239,  247 
Molecular  movement,  23 
Monas,  8 

Monomorphism.  19 
Monotrichic,  *24 
Morphin,  102 
Morphology,  *15-21 
Motility,  23 
Mucor,  *31,  37 
Mulching,  249,  254,  265 
Multiplication,  25 
Mycelium,  26 


Mycobacteriaceae,  38 
My co derma,  37 
Mycorrhiza,  113 
Myrica,  113 
Myxobacteriaceae,  223 
Mvxomycetes,  223 

N 

Neufchatel  cheese,  205,  206 
Nicotin,  102 
Niter  spots,  254 
Nitragin,  272 

Nitrate  assimilation,  *43,  105,  254 
formation,  102-104,  253 
reduction,  104,  105 

Nitrification,  46,  102-104,  *108,  231,  248, 
253 

Nitrobacter,  103 
Nitrobacterine,  272 
Nitroculture,  272 

Nitrogen  assimilation,  *108-117,  233 
availability,  224,  254 
-carbon  ratio,  94,  128 
content,  40 
cycle,  *95 

fixation,  109-117,  233,  *256-262,  *270- 
272 

gains,  116,  258,  261 
liberation,  106-108,  233 
losses,  232,  234,  255 
metabolism,  *95,  228-233,  250 
oxidation,  99 
requirement,  41 
Nitrosococcus,  103 
Nitrosomonas,  *103 
Niveaux,  28,  *135 

Nodule  bacteria,  *18,  111,  259,  *260 

Nomenclature,  35 

Nostoc,  113 

Nucleoproteids,  131 

Nucleus,  21 

Number  and  size  of  bacteria,  *17 
Nutrient  solutions,  69,  73,  239,  243 
Nutrition,  39 

O 

Oidia,  37 

Oidium  lactis,  *37,  121,  127,  176,  183,  205 
Oily  butter,  192 
Opsonin,  142 

Organic  manures,  245,  250-254,  264 


INDEX 


280 

Oxalic  acid,  127 
Oxidation,  49 
Oxygen,  47-49,  117 
Ozone,  84 

> 

Paper,  germ  content  of,  189 
Para-phenylen-diamin,  184 
Parasites,  39,  138 
Parmesan  cheese,  214 
Pasteurization,  78,  170,  195,  212 
Pathogenic  organisms,  138-145 
in  butter,  191 
in  cheese,  198 
in  digestive  tract,  159 
in  manure,  215,  224 
in  milk,  162,  171 
in  water,  220 
Pavetta,  113 
Pea  nodules,  *256 
Peat,  114,  128,  216,  231,  232,  253,  265 
Pectin  decomposition,  124,  225 
Penicillium,  *28,  *31,  38,  204,  205 
Pepsin,  204 
Pppton,  *43,  *101 
Perithecia,  38 
Peritrichic,  *24 
Permanganate,  84,  85 
Peroxide  of  hydrogen,  84 
Petri  dish,  *27 
Phagocytes,  *141,  167 
Phenol,  85,  231,  268 
Phoma,  113 

Phosphates,  131,  132,  235 
Phosphatides,  131 
Phosphogerm,  271 
Phosphorescent  bacteria,  *90 
Phosphorus,  42 

metabolism,  131,  224 
Physical  factors,  44 
Phytin,  131 

Pigment  production,  *88,  182,  194 
Pink  yeast,  *88 

Plant  growth  and  soil  flora,  266 
Plant  inoculation,  *271,  *272 
Plants,  germ  content  of,  62,  1 49 
Plasmolysis,  22 
Plasmoptysis,  22 
Plate  cultures,  69 
Plectridium,  *30 
pectinovorum,  124 


Pleomorphism,  19 
Podocarpus,  113 
Polymorphism,  19 
Pot  experiments,  77,  243 
Potassium,  133 
metabolism,  224 
permanganate,  84 
Potato  bacilli,  59 
Potatoes,  germ  content  of,  150 
spoilage  of,  *158 
storage  of,  158 
Preserving  food,  8 
Privy,  216 

Propionic  acid,  127,  154,  202 
Protein  decomposition,  98 
Proteus,  *18 
Protista,  38 
Protoplasm,  *21 
Prototrophic,  40 
Protozoa,  38 
cysts,  32 
motility,  38 
multiplication,  26 
presence  in  manure,  223 
presence  in  soil,  238,  239,  242,  264,  269 
sporozoites,  32 
Pseudomonas,  37 
Pseudopodia,  38 
Psychotria.  113 
Psychrophilic,  51 
Ptomains,  139 
Puma,  122,  187 
Pulp  cultures,  *156 
Pure  cultures,  54,  74 
Purple  bacteria,  88,  136 
Putrefaction,  8,  94 
Putrescin,  205 
Pyrogallic  acid,  72 

Q 

Quinin,  102 

R 

Radium  rays,  53 
Rancid  fats,  192-194,  206 
milk,  181 

Rape  cake,  45,  252 
Reaction,  44 
Reduction,  49 

test,  *172-174,  1S5 
Refrigeration,  169 


INDEX 


281 


Regeneration,  *29- *33 
Regenerative  bodies,  *31,  *32 
units,  *32 

Relativity  of  action,  87 
Rennet,  198,  200,  208,  210 
Rennet  producing  bacteria,  180 
Reproductive  organs,  *29- *32,  58-60 
Resistance,  47,  51,  58-60,  79,  83-85,  138 
Respiration,  47,  50 
Resting  forms,  29-32,  58-60 
Retting  of  flax,  124 
Rhizobium,  112 
Rhizofication,  98 
Rhizopoda,  38 
Ripening  centers,  199 
Ripening  of  cheese,  201 
of  cream,  196 
of  manure,  225 
of  milk,  186,  211 
of  soil,  250 

Rock  phosphates,  132 
Roentgen  rays,  53 
Root  excretions,  245 
nodules,  110,  *256,  *257 
Roots,  activity  of,  50 
Ropy  milk,  183 

Roquefort  cheese,  203-205,  214 
Rotting  of  manure,  106,  224-236,  251,  252 
Roughage,  germ  content  of,  68,  166 
Ruminants,  intestinal  flora  of,  63,  186 
Rusty  containers,  182 

S 

Saccharomycetes,  37 

Salicylic  acid,  85 

Salt,  84,  152,  155,  157,  189,  211 

Saltpeter,  212 

Sand  filter,  *80 

Saprophytes,  39 

Sarcina,  *20,  35 

Sarcodina,  38 

Sauerkraut,  157 

Sawdust,  232 

Scarlet  fever,  171 

Schizomycetes,  25 

Scouring,  82 

Scrubbing,  82 

Seasonal  effects,  238,  *240 

Self-purification  of  water,  56 

Septic  tanks,  *217 

Serradella,  *270 


Serum  therapy,  144 
Sewage  disposal,  215-220 
Sexual  processes,  *29 
Sheath,  22 
Sheep  manure,  230 
Silage,  152,  *154 
Silicates,  132 
Single-cell  cultures,  *70 
Size  of  microorganisms,  *15-17 
Slime  production,  *22,  121,  183 
Soda,  caustic,  83 
Sodium  hydrosulfite,  72 
Soft  cheeses,  197 
Soil  acidity,  128,  249,  253 
aeration,  238,  245,  263 
atmosphere,  246 
cultivation,  262-272 
disinfection,  *267 
extract,  243 

fertility,  114,  247,  258,  264 
germ  content,  *60,  237-272 
inoculation,  *270-272 
irrigation,  263 

moisture,  45,  243,  255,  263,  267 

mulching,  249,  254,  265 

nitrogen  content,  247-249 

productivity,  114,  237,  241,  264 

reaction,  266 

ripening,  250 

sickness,  242 

sterilization,  *267 

tillage,  53,  242,  2G3 

tilth,  249,  262 

water  content,  46,  242,  253,  255,  263-267 
Soy  bean  nodules,  *256 
Soyka  flasks,  *27 
Spathodea,  113 
Specific  weight,  40 
Speed  of  bacteria,  23 
Spirillaceae,  37 
Spirillum,  36 
Spirochaeta,  36 
Spoilage  of  food,  157 
Spontaneous  generation,  9 
ignition,  92 
Sporangia,  *31 
Spores,  *29-32 
Sporozoites,  32 
Spring  maxima,  238,  *240 
Stab  cultures,  *46,  *72 
Stable  manure  (see  Manure). 


INDEX 


282 


Staining  methods,  76 
Staphylococcus,  *20 
Starch,  118,  246 
Starters,  *196,  *213 
Steam,  59,  79,  165 
Sterilization,  *73,  *74,  78,  164 
Stilton  cheese,  204 
Stimulants,  43,  269 
Stomach,  germ  content  of,  63,  186 
Straw,  effect  in  soil,  254,  255,  264 
germ  content  of,  150,  222,  232 
rotting  of,  128 
Streptococci  in  milk,  *18 
Streptococcus,  35 
acidi  paralactici,  35 
hollandicus,  122 
■ lactis,  58,  120 
pyogenes,  121,  162 
Strychin,  102 
Sub  irrigation,  218 
Sublimate,  84 
Subsoil,  238,  242 
Substrates,  73 
Succinic  acid,  127,  179 
Sulfate  formation,  *135 
reduction,  136 
Sulfofication,  135 
Sulfur,  133,  236 
bacteria,  *136 
effect  in  soil,  266 
metabolism,  *133 
Sugar,  effect  in  soil,  246,  *262 
fermentation,  118 
Sunflower  silage,  153,  154 
Sunlight,  52,  182 
Surface  effect,  *17 
Swamps,  105 
Swedish  hard  cheese,  204 
Swiss  cheese,  197,  204,  *209,  213 
Symbiosis,  54,  *57,  205,  261 
Symplasm,  *32 
System,  34-38 

T 

Taettemjolk,  122,  187 
Tallowy  butter,  194 
taste  of  milk,  182 
Tartaric  acid,  127 
Temperature,  51,  59 
influence  upon  butter,  193 
upon  cheese,  198,  199 


Temperature,  influence  on  manure,  226 
upon  milk,  167 
Terminology,  97 
Testing  pure  cultures,  74 
Thermogenic,  51 
Thermophilic,  51 
Thiobacillus  denitrificans,  135 
thiooxidans,  136 
thioparus,  136 
Thiosulfates,  134 
Thread  formation,  *20 
Thunderstorms,  181 
Tilth,  249,  262 
Timothy  bacillus,  150 
Tobacco  fermentation,  92 
Toluol,  267 
Torula,  37 

Toxins,  102,  139,  208 
Transporting  milk,  168 
Trickling  filters,  *219 
Trimethylamin,  192 
Tubercle  bacilli,  171,  191 
Typhoid,  171,  216 
Tyrosin,  99 
Tyrothrix,  204 

U 

U-cultures,  271 

Udder  bacteria,  161-163,  181,  198 
Ultra-microorganisms,  16 
Ultra-violet  rays,  53,  80 
Urea,  99,  *101 
bacteria,  *8,  100 
Urease,  100 
Uric  acid,  100 
Urine,  222,  229-231 

Utensils,  germ  content  of,  165,  181  -183, 
189,  212 

V 

Vaccination,  143 
Vacuoles,  22 

Variability  of  action,  65,  90 
of  cell  form,  *18 
of  colony  formation,  28 
Vibrio,  36 

Virgin  soils,  247,  249 
Virulence,  86,  140 
Volutin,  40 

W 

Water,  germ  content  of,  62,  165 
purification,  *S0,  217-220 


INDEX 


283 


Water,  requirement,  45 
sterilization,  84 

Water  content  of  bacterial  cells,  40 
of  butter,  188 
of  cheese,  197 
of  food,  45 
of  soil,  46,  242,  253 
Water-holding  capacity,  46,  264 
Wensleydale  cheese,  204 
Westrobac,  272 
Whey,  180,  236 

Winter,  effect  on  soil  flora,  238,  240 
Wisconsin  curd  test,  175 
Worms  in  soil,  248 

X 

X-rays,  53 
Xylol,  268 


Y 

Yaourt,  *186 
Yeasts,  *8,  *21,  *27,  37 
alcohol  formation  of,  122,  155,  180 
in  butter,  191 
in  cheese,  79,  200,  207 
in  milk,  178,  180 
in  nitrate,  212 
in  sauerkraut,  157 
in  silage,  155 
Yeasty  taste,  192 
Yoghurt,  *186 

Z 

Zinc  salts,  268 
Zoogloea,  22 
Zygospores,  *31 


I 


J 


589.95  L825T 


281124 


£,fv  tj.  \ f'* 


&pT  _ uiEKARY 


